Pyrimidine hydroxy amide compounds as protein deacetylase inhibitors and methods of use thereof

ABSTRACT

The present invention relates to novel pyrimidine hydroxy amide compounds, and the use of such compounds in the inhibition of HDAC6 and in the treatment of various diseases, disorders or conditions related to HDAC6.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application61/414,158, filed on Nov. 16, 2010, and U.S. provisional application61/503,286, filed on Jun. 30, 2011. The entire contents of theaforementioned applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The identification of small organic molecules that affect specificbiological functions is an endeavor that impacts both biology andmedicine. Such molecules are useful as therapeutic agents and as probesof biological function. Such small molecules have been useful atelucidating signal transduction pathways by acting as chemical proteinknockouts, thereby causing a loss of protein function. (Schreiber et al,J. Am. Chem. Soc, 1990, 112, 5583; Mitchison, Chem. and Biol., 1994, I53) Additionally, due to the interaction of these small molecules withparticular biological targets and their ability to affect specificbiological function (e.g. gene transcription), they may also serve ascandidates for the development of new therapeutics.

One biological target of recent interest is histone deacetylase (HDAC)(see, for example, a discussion of the use of inhibitors of histonedeacetylases for the treatment of cancer: Marks et al. Nature ReviewsCancer 2001, 7,194; Johnstone et al. Nature Reviews Drug Discovery 2002,287). Post-translational modification of proteins through acetylationand deacetylation of lysine residues plays a critical role in regulatingtheir cellular functions. HDACs are zinc hydrolases that modulate geneexpression through deacetylation of the N-acetyl-lysine residues ofhistone proteins and other transcriptional regulators (Hassig et alCurr. Opin. Chem. Biol. 1997, 1, 300-308). HDACs participate in cellularpathways that control cell shape and differentiation, and an HDACinhibitor has been shown effective in treating an otherwise recalcitrantcancer (Warrell et al J. Natl. Cancer Inst. 1998, 90, 1621-1625). Atthis time, eleven human HDACs, which use Zn as a cofactor, have beenidentified (Taunton et al. Science 1996, 272, 408-411; Yang et al. J.Biol. Chem. 1997, 272, 28001-28007. Grozinger et al. Proc. Natl. Acad.Sci. U.S.A. 1999, 96, 4868-4873; Kao et al. Genes Dev. 2000, 14, 55-66.Hu et al J. Biol. Chem. 2000, 275, 15254-15264; Zhou et al. Proc. Natl.Acad. Sci. U.S.A. 2001, 98, 10572-10577; Venter et al. Science 2001,291, 1304-1351) these members fall into three classes (class I, II, andIV). An additional seven HDACs have been identified which use NAD as acofactor. To date, no small molecules are known that selectively targetany particular class or individual members of this family (for exampleortholog-selective HDAC inhibitors have been reported: (a) Meinke et al.J. Med. Chem. 2000, 14, 4919-4922; (b) Meinke, et al Curr. Med. Chem.2001, 8, 211-235). There remains a need for preparing structurallydiverse HDAC and tubulin deacetylase (TDAC) inhibitors particularly onesthat are potent and/or selective inhibitors of particular classes ofHDACs or TDACs and individual HDACs and TDACs.

Recently, a cytoplasmic histone deacetylase protein, HDAC6, wasidentified as necessary for aggresome formation and for survival ofcells following ubiquitinated misfolded protein stress. The aggresome isan integral component of survival in cancer cells. The mechanism ofHDAC6-mediated aggresome formation is a consequence of the catalyticactivity of the carboxy-terminal deacetylase domain, targeting anuncharacterized non-histone target. The present invention also providessmall molecule inhibitors of HDAC6. In certain embodiments, these newcompounds are potent and selective inhibitors of HDAC6.

The aggresome was first described in 1998, when it was reported thatthere was an appearance of microtubule-associated perinuclear inclusionbodies in cells over-expressing the pathologic ΔF508 allele of thecystic fibrosis transmembrane conductance receptor (CFTR). Subsequentreports identified a pathologic appearance of the aggresome withover-expressed presenilin-1 (Johnston J A, et al. J Cell Biol. 1998;143:1883-1898), parkin (Junn E, et al. J Biol Chem. 2002; 277:47870-47877), peripheral myelin protein PMP22 (Notterpek L, et al.Neurobiol Dis. 1999; 6: 450-460), influenza virus nucleoprotein (Anton LC, et al. J Cell Biol. 1999; 146:113-124), a chimera of GFP and themembrane transport protein pi 15 (Garcia-Mata R, et al. J Cell Biol.1999; 146: 1239-1254) and notably amyloidogenic light chains (Dui J L,et al. J Cell Biol. 2001; 152:705-716). Model systems have beenestablished to study ubiquitinated (ΔF508 CFTR) (Johnston J A, et al. JCell Biol. 1998; 143:1883-1898) and non-ubiquitinated (GFP-250)(Garcia-Mata R, et al. J Cell Biol. 1999; 146:1239-1254) proteinaggregate transport to the aggresome. Secretory, mutated, and wild-typeproteins may assume unstable kinetic intermediates resulting in stableaggregates incapable of degradation through the narrow channel of the26S proteasome. These complexes undergo active, retrograde transport bydynein to the pericentriolar aggresome, mediated in part by acytoplasmic histone deacetylase, HDAC6 (Kawaguchi Y, et al. Cell. 2003;115:727-738).

Histone deacetylases are a family of at least 11 zinc-bindinghydrolases, which catalyze the deacetylation of lysine residues onhistone proteins. HDAC inhibition results in hyperacetylation ofchromatin, alterations in transcription, growth arrest, and apoptosis incancer cell lines. Early phase clinical trials with availablenonselective HDAC inhibitors demonstrate responses in hematologicmalignancies including multiple myeloma, although with significanttoxicity. Of note, in vitro synergy of conventional chemotherapy agents(such as melphalan) with bortezomib has been reported in myeloma celllines, though dual proteasome-aggresome inhibition was not proposed.Until recently selective HDAC inhibitors have not been realized.

HDAC6 is required for aggresome formation with ubiquitinated proteinstress and is essential for cellular viability in this context. HDAC6 isbelieved to bind ubiquitinated proteins through a zinc finger domain andinteracts with the dynein motor complex through another discrete bindingmotif. HDAC6 possesses two catalytic deacetylase domains. It is notpresently known whether the amino-terminal histone deacetylase or thecarboxy-terminal tubulin deacetylase (TDAC) domain mediates aggresomeformation.

Aberrant protein catabolism is a hallmark of cancer, and is implicatedin the stabilization of oncogenic proteins and the degradation of tumorsuppressors (Adams J. Nat Rev Cancer. 2004; 4:349-360). Tumor necrosisfactor alpha induced activation of nuclear factor kappa B (NFKB) is arelevant example, mediated by NFKB inhibitor beta (IKB) proteolyticdegradation in malignant plasma cells. The inhibition of IKB catabolismby proteasome inhibitors explains, in part, the apoptotic growth arrestof treated myeloma cells (Hideshima T, et al. Cancer Res. 2001;61:3071-3076). Multiple myeloma is an ideal system for studying themechanisms of protein degradation in cancer. Since William Russell in1890, cytoplasmic inclusions have been regarded as a defininghistological feature of malignant plasma cells. Though the precisecomposition of Russell bodies is not known, they are regarded asER-derived vesicles containing aggregates of monotypic immunoglobulins(Kopito R R, Sitia R. EMBO Rep. 2000: 1:225-231) and stain positive forubiquitin (Manetto V, et al. Am J Pathol. 1989; 134:505-513). Russellbodies have been described with CFTR over-expression in yeast (SullivanM L, et al. J. Histochem. Cytochem. 2003; 51:545-548), thus raising thesuspicion that these structures may be linked to overwhelmed proteincatabolism, and potentially the aggresome. The role of the aggresome incancer remains undefined.

Aberrant histone deacetylase activity has also been linked to variousneurological and neurodegenerative disorders, including stroke,Huntington's disease, Amyotrophic Lateral Sclerosis and Alzheimer'sdisease. HDAC inhibition may induce the expression of anti-mitotic andanti-apoptotic genes, such as p21 and HSP-70, which facilitate survival.HDAC inhibitors can act on other neural cell types in the centralnervous system, such as reactive astrocytes and microglia, to reduceinflammation and secondary damage during neuronal injury or disease.HDAC inhibition is a promising therapeutic approach for the treatment ofa range of central nervous system disorders (Langley B et al., 2005,Current Drug Targets—CNS & Neurological Disorders, 4: 41-50).

Histone deacetylase is known to play an essential role in thetranscriptional machinery for regulating gene expression, induce histonehyperacetylation and to affect the gene expression. Therefore, it isuseful as a therapeutic or prophylactic agent for diseases caused byabnormal gene expression such as inflammatory disorders, diabetes,diabetic complications, homozygous thalassemia, fibrosis, cirrhosis,acute promyelocytic leukaemia (APL), organ transplant rejections,autoimmune diseases, protozoal infections, tumors, etc. Thus, thereremains a need for the development of novel inhibitors of histonedeacetylases and tubulin histone deacetylases. In particular, inhibitorsthat are more potent and/or more specific for their particular targetthan known HDAC and TDAC inhibitors are needed. HDAC inhibitors specificfor a certain class or member of the HDAC family would be particularlyuseful both in the treatment of proliferative diseases and proteindeposition disorders and in the study of HDACs, particularly HDAC6.Inhibitors that are specific for HDAC versus TDAC and vice versa arealso useful in treating disease and probing biological pathways. Thepresent invention provides novel compounds, pharmaceutical compositionsthereof, and methods of using these compounds to treat disorders relatedto HDAC6 including cancers, inflammatory, autoimmune, neurological andneurodegenerative disorders.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a compound of formula I:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

each of X₁, X₂, X₃, or X₄ is independently N or CR′, wherein 2 of X₁,X₂, X₃, or X₄ are N;

each R′ is independently H, optionally substituted alkyl, optionallysubstituted aryl, or halo:

R₁ is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted; and R₂ is H, alkyl, haloalkyl,alkenyl, arylalkyl, aryl, heteroaryl, heterocyclic, carbocyclic,—C(O)R₅, or —S(O)R₅, each of which may be optionally substituted;

p is 0, 1, or 2;

each R₅ is independently H; optionally substituted alkyl, optionallysubstituted carbocyclic, optionally substituted heterocyclic, optionallysubstituted aryl, or optionally substituted heteroaryl;

wherein R₁ and R₂ are both not simultaneously H.

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound of any of the formulae herein (e.g., formula I).

In another aspect, the invention provides a method of selectivelyinhibiting HDAC6 over other HDACs in a subject, comprising administeringto the subject a compound of any of the formulae herein (e.g., formulaI).

In another aspect, the invention provides a method of treating a diseasemediated by HDAC6 in a subject comprising administering to the subject acompound of any of the formulae herein (e.g., formula I).

In another aspect, the invention provides a method of treating a subjectsuffering from or susceptible to multiple myeloma comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound of any of the formulae herein (e.g., formula I), tothereby treat the subject suffering from or susceptible to multiplemyeloma.

In another aspect, the invention provides a kit comprising a compoundcapable of inhibiting HDAC activity selected from one or more compoundsof any of the formulae herein (e.g., formula I); and instructions foruse in treating multiple myeloma.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.

The number of carbon atoms in a hydrocarbyl substituent can be indicatedby the prefix “C_(x)-C_(y),” where x is the minimum and y is the maximumnumber of carbon atoms in the substituent. Likewise, a C_(x) chain meansa hydrocarbyl chain containing x carbon atoms.

If a linking element in a depicted structure is “absent” or a “bond”,then the left element in the depicted structure is directly linked tothe right element in the depicted structure. For example, if a chemicalstructure is depicted as X-(L)_(n)-Y wherein L is absent or n is 0, thenthe chemical structure is X—Y.

The term “alkyl,” as used herein, refers to saturated, straight- orbranched-chain hydrocarbon moieties containing, in certain embodiments,between one and six, or one and eight carbon atoms, respectively.Examples of C₁-C₆ alkyl moieties include, but are not limited to,methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl,n-hexyl moieties; and examples of C₁-C₈ alkyl moieties include, but arenot limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl,neopentyl, n-hexyl, heptyl, and octyl moieties.

The term “alkenyl,” as used herein, denotes a monovalent group derivedfrom a hydrocarbon moiety containing, in certain embodiments, from twoto six, or two to eight carbon atoms having at least one carbon-carbondouble bond. The double bond may or may not be the point of attachmentto another group. Alkenyl groups include, but are not limited to, forexample, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl,octenyl and the like.

The term “alkynyl,” as used herein, denotes a monovalent group derivedfrom a hydrocarbon moiety containing, in certain embodiments, from twoto six, or two to eight carbon atoms having at least one carbon-carbontriple bond. The alkynyl group may or may not be the point of attachmentto another group. Representative alkynyl groups include, but are notlimited to, for example, ethynyl, 1-propynyl, 1-butynyl, heptynyl,octynyl and the like.

The term “alkoxy” refers to an —O-alkyl moiety.

The term “aryl,” as used herein, refers to a mono- or poly-cycliccarbocyclic ring system having one or more aromatic rings, fused ornon-fused, including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, idenyl and the like.

The term “aralkyl,” or “arylalkyl,” as used herein, refers to an alkylresidue attached to an aryl ring. Examples include, but are not limitedto, benzyl, phenethyl and the like.

The term “carbocyclic,” as used herein, denotes a monovalent groupderived from a monocyclic or polycyclic saturated, partially unsatured,or fully unsaturated carbocyclic ring compound. Examples of carbocyclicgroups include groups found in the cycloalkyl definition and aryldefinition.

The term “cycloalkyl,” as used herein, denotes a monovalent groupderived from a monocyclic or polycyclic saturated or partially unsaturedcarbocyclic ring compound. Examples of C₃-C₈-cycloalkyl include, but notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cyclopentyl and cyclooctyl; and examples of C₃-C₁₂-cycloalkyl include,but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,bicyclo [2.2.1] heptyl, and bicyclo [2.2.2] octyl. Also contemplated aremonovalent groups derived from a monocyclic or polycyclic carbocyclicring compound having at least one carbon-carbon double bond by theremoval of a single hydrogen atom. Examples of such groups include, butare not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like.

The term “heteroaryl,” as used herein, refers to a mono- or poly-cyclic(e.g., bi-, or tri-cyclic or more) fused or non-fused, moieties or ringsystem having at least one aromatic ring, having from five to ten ringatoms of which one ring atom is selected from S, O and N; zero, one ortwo ring atoms are additional heteroatoms independently selected from S,O and N; and the remaining ring atoms are carbon. Heteroaryl includes,but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.

The term “heteroaralkyl,” as used herein, refers to an alkyl residueresidue attached to a heteroaryl ring. Examples include, but are notlimited to, pyridinylmethyl, pyrimidinylethyl and the like.

The term “heterocycloalkyl,” as used herein, refers to a non-aromatic3-, 4-, 5-, 6- or 7-membered ring or a bi- or tri-cyclic group fused ofnon-fused system, where (i) each ring contains between one and threeheteroatoms independently selected from oxygen, sulfur and nitrogen,(ii) each 5-membered ring has 0 to 1 double bonds and each 6-memberedring has 0 to 2 double bonds, (iii) the nitrogen and sulfur heteroatomsmay optionally be oxidized, (iv) the nitrogen heteroatom may optionallybe quaternized, and (iv) any of the above rings may be fused to abenzene ring. Representative heterocycloalkyl groups include, but arenot limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, and tetrahydrofuryl.

The term “alkylamino” refers to a group having the structure —NH(C₁-C₁₂alkyl) where C₁-C₁₂ alkyl is as previously defined.

The term “acyl” includes residues derived from acids, including but notlimited to carboxylic acids, carbamic acids, carbonic acids, sulfonicacids, and phosphorous acids. Examples include aliphatic carbonyls,aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphaticsulfinyls, aromatic phosphates and aliphatic phosphates. Examples ofaliphatic carbonyls include, but are not limited to, acetyl, propionyl,2-fluoroacetyl, butyryl, 2-hydroxy acetyl, and the like.

In accordance with the invention, any of the aryls, substituted aryls,heteroaryls and substituted heteroaryls described herein, can be anyaromatic group. Aromatic groups can be substituted or unsubstituted.

The terms “hal,” “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The term “oxo” as used herein, refers to an oxygen that is attached to acarbon, preferably by a double bond (e.g., carbonyl).

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as are illustratedgenerally above, or as exemplified by particular classes, subclasses,and species of the invention. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted”,whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. Unless otherwise indicated, an optionallysubstituted group may have a substituent at each substitutable positionof the group, and when more than one position in any given structure maybe substituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. The terms “optionally substituted”, “optionally substitutedalkyl,” “optionally substituted” “optionally substituted alkenyl,”“optionally substituted alkynyl”, “optionally substituted cycloalkyl,”“optionally substituted cycloalkenyl,” “optionally substituted aryl”,“optionally substituted heteroaryl,” “optionally substituted aralkyl”,“optionally substituted heteroaralkyl,” “optionally substitutedheterocycloalkyl,” and any other optionally substituted group as usedherein, refer to groups that are substituted or unsubstituted byindependent replacement of one, two, or three or more of the hydrogenatoms thereon with substituents including, but not limited to:

alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl,arylalkyl, heteroarylalkyl, haloalkyl (e.g., —CF₃), haloalkoxy (e.g.,—OCF₁),

—F, —Cl, —Br, —I,

—OH, protected hydroxy, oxygen, oxo,

—NO₂, —CN,

—NH₂, protected amino, —NH—C₁-C₁₂-alkyl, —NH-aryl, -dialkylamino,

—O—C₁-C₁₂-alkyl, —O-aryl,

—C(O)—, —C(O)O—, —C(O)NH—, —OC(O)—, —OC(O)O—, —OC(O)NH—, —NHC(O)—,—NHC(O)O—, —C(O)—C₁-C₁₂-alkyl, —C(O)—C₃-C₁₂-cycloalkyl, —C(O)-aryl,—C(O)-heteroaryl, —C(O)-heterocycloalkyl,

—C(O)O—C₁-C₁₂-alkyl, —C(O)O—C₃-C₁₂-cycloalkyl, —C(O)O-aryl,—C(O)O-heteroaryl, —C(O)O-heterocycloalkyl,

—CONH₂, —CONH—C₁-C₁₂-alkyl, —CONH-aryl,

—OCO₂—C₁-C₁₂-alkyl, —OCO₂-aryl, —OCONH₂, —OCONH—C₁-C₁₂-alkyl,—OCONH-aryl,

—NHC(O)—C₁-C₁₂-alkyl, —NHC(O)-aryl, —NHCO₂—C₁-C₁₂-alkyl, —NHCO₂-aryl,

-   -   —S(O)—C₁-C₁₂-alkyl, —S(O)-aryl, —SO₂NH—C₁-C₁₂-alkyl,        —SO₂NH-aryl,

—NHSO₂—C₁-C₁₂-alkyl, —NHSO₂-aryl, or

—SH, —S—C₁-C₁₂-alkyl, or —S-aryl.

In certain embodiments, the optionally substituted groups include thefollowing: C₁-C₁₂-alkyl, C₂-C₁₂-alkenyl, C₂-C₁₂-alkynyl,C₃-C₁₂-cycloalkyl, C₃-C₁₂-aryl, C₃-C₁₂-heterocycloalkyl,C₃-C₁₂-heteroaryl, C₄-C₁₂-arylalkyl, or C₂-C₁₂-heteroarylalkyl.

It is understood that the aryls, heteroaryls, alkyls, and the like canbe further substituted.

As used herein, the term “metal chelator” refers to any molecule ormoiety that is capable of forming a complex (i.e., “chelates”) with ametal ion. In certain exemplary embodiments, a metal chelator refers toto any molecule or moiety that “binds” to a metal ion, in solution,making it unavailable for use in chemical/enzymatic reactions. Incertain embodiments, the solution comprises aqueous environments underphysiological conditions. Examples of metal ions include, but are notlimited to, Ca²⁺, Fe³⁺, Zn²⁺, Na⁺, etc. In certain embodiments, themetal chelator binds Zn²⁺. In certain embodiments, molecules of moietiesthat precipitate metal ions are not considered to be metal chelators.

As used herein, the term “small molecule” refers to a non-peptidic,non-oligomeric organic compound either synthesized in the laboratory orfound in nature. Small molecules, as used herein, can refer to compoundsthat are “natural product-like”, however, the term “small molecule” isnot limited to “natural product-like” compounds. Rather, a smallmolecule is typically characterized in that it contains severalcarbon-carbon bonds, and has a molecular weight of less than 1500,although this characterization is not intended to be limiting for thepurposes of the present invention. Examples of “small molecules” thatoccur in nature include, but are not limited to, taxol, dynemicin, andrapamycin. In certain other preferred embodiments, natural-product-likesmall molecules are utilized.

The term “subject” as used herein refers to a mammal. A subjecttherefore refers to, for example, dogs, cats, horses, cows, pigs, guineapigs, and the like. Preferably the subject is a human. When the subjectis a human, the subject may be referred to herein as a patient.

“Treat”, “treating” and “treatment” refer to a method of alleviating orabating a disease and/or its attendant symptoms.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts of the compounds formed by the process of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared insitu during the final isolation and purification of the compounds of theinvention, or separately by reacting the free base function with asuitable organic acid. Examples of pharmaceutically acceptable include,but are not limited to, nontoxic acid addition salts are salts of anamino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, maleic acid, tartaric acid,citric acid, succinic acid or malonic acid or by using other methodsused in the art such as ion exchange. Other pharmaceutically acceptablesalts include, but are not limited to, adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and arylsulfonate.

As used herein, the term “pharmaceutically acceptable ester” refers toesters of the compounds formed by the process of the present inventionwhich hydrolyze in vivo and include those that break down readily in thehuman body to leave the parent compound or a salt thereof. Suitableester groups include, for example, those derived from pharmaceuticallyacceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic,cycloalkanoic and alkanedioic acids, in which each alkyl or alkenylmoiety advantageously has not more than 6 carbon atoms. Examples ofparticular esters include, but are not limited to, formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

The term “pharmaceutically acceptable prodrugs” as used herein refers tothose prodrugs of the compounds formed by the process of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the present invention. “Prodrug”, as used hereinmeans a compound which is convertible in vivo by metabolic means (e.g.by hydrolysis) to afford any compound delineated by the formulae of theinstant invention. Various forms of prodrugs are known in the art, forexample, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier(1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4, AcademicPress (1985); Krogsgaard-Larsen, et al., (ed). “Design and Applicationof Prodrugs, Textbook of Drug Design and Development, Chapter 5, 113-191(1991); Bundgaard, et al., Journal of Drug Deliver Reviews,8:1-38(1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq.(1988); Higuchi and Stella (eds.) Prodrugs as Novel Drug DeliverySystems, American Chemical Society (1975); and Bernard Testa & JoachimMayer, “Hydrolysis In Drug And Prodrug Metabolism: Chemistry,Biochemistry And Enzymology,” John Wiley and Sons, Ltd. (2002).

This invention also encompasses pharmaceutical compositions containing,and methods of treating disorders through administering,pharmaceutically acceptable prodrugs of compounds of the invention. Forexample, compounds of the invention having free amino, amido, hydroxy orcarboxylic groups can be converted into prodrugs. Prodrugs includecompounds wherein an amino acid residue, or a polypeptide chain of twoor more (e.g., two, three or four) amino acid residues is covalentlyjoined through an amide or ester bond to a free amino, hydroxy orcarboxylic acid group of compounds of the invention. The amino acidresidues include but are not limited to the 20 naturally occurring aminoacids commonly designated by three letter symbols and also includes4-hydroxyproline, hydroxyysine, demosine, isodemosine,3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid,citrulline, homocysteine, homoserine, ornithine and methionine sulfone.Additional types of prodrugs are also encompassed. For instance, freecarboxyl groups can be derivatized as amides or alkyl esters. Freehydroxy groups may be derivatized using groups including but not limitedto hemisuccinates, phosphate esters, dimethylaminoacetates, andphosphoryloxymethyloxy carbonyls, as outlined in Advanced Drug DeliveryReviews, 1996, 19, 1 15. Carbamate prodrugs of hydroxy and amino groupsare also included, as are carbonate prodrugs, sulfonate esters andsulfate esters of hydroxy groups. Derivatization of hydroxy groups as(acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group may bean alkyl ester, optionally substituted with groups including but notlimited to ether, amine and carboxylic acid functionalities, or wherethe acyl group is an amino acid ester as described above, are alsoencompassed. Prodrugs of this type are described in J. Med. Chem. 1996,39, 10. Free amines can also be derivatized as amides, sulfonamides orphosphonamides. All of these prodrug moieties may incorporate groupsincluding but not limited to ether, amine and carboxylic acidfunctionalities

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable”, as used herein, refers to compounds which possessstability sufficient to allow manufacture and which maintains theintegrity of the compound for a sufficient period of time to be usefulfor the purposes detailed herein (e.g., therapeutic or prophylacticadministration to a subject).

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. Particularly, in embodiments thecompound is at least 85% pure, more preferably at least 90% pure, morepreferably at least 95% pure, and most preferably at least 99% pure.

As used herein, the term “disease” includes any one or more of thefollowing autoimmune diseases or disorders: diabetes mellitus, arthritis(including rheumatoid arthritis, juvenile rheumatoid arthritis,osteoarthritis, psoriatic arthritis), multiple sclerosis, myastheniagravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis(including atopic dermatitis and eczematous dermatitis), psoriasis,Sjögren's Syndrome, including keratoconjunctivitis sicca secondary toSjögren's Syndrome, alopecia areata, allergic responses due to arthropodbite reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drugeruptions, leprosy reversal reactions, erythema nodosum leprosum,autoimmune uveitis, allergic encephalomyelitis, acute necrotizinghemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis. Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Graves ophthalmopathy, sarcoidosis, primary biliarycirrhosis, uveitis posterior, and interstitial lung fibrosis.

In another embodiment, disease refers to any one of Wilson's disease,spinocerebellar ataxia, prion disease, Parkinson's disease, Huntington'sdisease, amytrophic lateral sclerosis, amyloidosis, Alzheimer's disease,Alexander's disease, alcoholic liver disease, cystic fibrosis, Pick'sDisease, spinal muscular dystrophy or Lewy body dementia.

“Disease” also includes any one of rheumatoid spondylitis; post ischemicperfusion injury; inflammatory bowel disease; chronic inflammatorypulmonary disease, eczema, asthma, ischemia/reperfusion injury, acuterespiratory distress syndrome, infectious arthritis, progressive chronicarthritis, deforming arthritis, traumatic arthritis, gouty arthritis,Reiter's syndrome, acute synovitis and spondylitis, glomerulonephritis,hemolytic anemia, aplastic anemia, neutropenia, host versus graftdisease, allograft rejection, chronic thyroiditis, Graves' disease,primary binary cirrhosis, contact dermatitis, skin sunburns, chronicrenal insufficiency, Guillain-Barre syndrome, uveitis, otitis media,periodontal disease, pulmonary interstitial fibrosis, bronchitis,rhinitis, sinusitis, pneumoconiosis, pulmonary insufficiency syndrome,pulmonary emphysema, pulmonary fibrosis, silicosis, or chronicinflammatory pulmonary disease.

“Disease” also refers to any one of cancer, tumor growth, cancer of thecolon, breast, bone, brain and others (e.g., osteosarcoma,neuroblastoma, colon adenocarcinoma), chronic myelogenous leukemia(CML), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL),cardiac cancer (e.g., sarcoma, myxoma, rhabdomyoma, fibroma, lipoma andteratoma); lung cancer (e.g., bronchogenic carcinoma, alveolarcarcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatoushamartoma, mesothelioma); various gastrointestinal cancer (e.g., cancersof esophagus, stomach, pancreas, small bowel, and large bowel);genitourinary tract cancer (e.g., kidney, bladder and urethra, prostate,testis; liver cancer (e.g., hepatoma, cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma); bonecancer (e.g., osteogenic sarcoma, fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma,multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma,benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteomaand giant cell tumors); cancers of the nervous system (e.g., of theskull, meninges, brain, and spinal cord); gynecological cancers (e.g.,uterus, cervix, ovaries, vulva, vagina); hematologic cancer (e.g.,cancers relating to blood, Hodgkin's disease, non-Hodgkin's lymphoma);skin cancer (e.g., malignant melanoma, basal cell carcinoma, squamouscell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma,angioma, dermatofibroma, keloids, psoriasis); and cancers of the adrenalglands (e.g., neuroblastoma).

Compounds of the Invention

In one aspect, the invention provides a compound of formula I:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

each of X₁, X₂, X₃, or X₄ is independently N or CR′, wherein 2 of X₁,X₂, X₃, or X₄ are N;

each R′ is independently H, optionally substituted alkyl, optionallysubstituted aryl, or halo;

R₁ is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted; and

R₂ is H, alkyl, haloalkyl, alkenyl, arylalkyl, aryl, heteroaryl,heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅, each of which may beoptionally substituted:

each p is independently 0, 1, or 2; and

each R₅ is independently H; optionally substituted alkyl, optionallysubstituted carbocyclic, optionally substituted heterocyclic, optionallysubstituted aryl, or optionally substituted heteroaryl;

wherein R₁ and R₂ are both not simultaneously H.

In one embodiment, the ring comprising X₁, X₂, X₃, and X₄ is selectedfrom the following:

In another embodiment, R₂ is H, methyl, ethyl, propyl, i-propyl, butyl,s-butyl, t-butyl, pentyl, hexyl, heptyl, phenyl, naphthyl, anthracenyl,pyridinyl, pyrimidinyl, pyrazinyl, indolyl, imidazolyl, oxazolyl, furyl,thienyl, thiophenyl, thiazolyl, triazolyl, isoxazolyl, quinolinyl,pyrrolyl, pyrazolyl, or 5,6,7,8-tetrahydroisoquinoline; each of whichmay be optionally substituted.

In another embodiment, R₁ is H, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted arylalkyl, optionallysubstituted heteroarylalkyl, or optionally substituted heteroaryl.

In a further embodiment, R₁ is H, methyl, ethyl, propyl, i-propyl,butyl, i-butyl, t-butyl, pentyl, hexyl, phenyl, benzyl, phenethyl,naphthyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, piperidinyl,pyrrolidinyl, tetrahydropyranyl, thiophenyl, or tetrahydrofuranyl, eachof which may be optionally substituted.

In certain embodiments, R₁ is substituted with 1-4 groups independentlyselected from optionally substituted alkyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substitutedheterocycloalkyl, halo, haloalkyl, alkoxy, haloalkoxy, and nitro.

In a further embodiment, R₁ is substituted with 1-4 groups independentlyselected from methyl, phenyl, pyridyl, morpholino, indolyl, pyrazinyl,F, Cl, Br, methoxy, OCF₃, and trifluoromethyl; each of which may befurther substituted.

In certain embodiments, each of the above substituents may be optionallysubstituted by alkyl, aryl, haloalkyl, haloalkoxy, halo, or alkoxy.

In another embodiment, R₂ is H.

In one embodiment, the invention provides a compound of formula II:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

each of X₁, X₂, or X₃, is independently N or CR′, wherein 2 of X₁, X₂,or X₃ are N;

R₂ is H, an optionally substituted alkyl, an optionally substituted arylor an optionally substituted heteroaryl;

R₁ is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, or carbocyclic, each of which may beoptionally substituted; and

each R′ is independently H, optionally substituted alkyl, optionallysubstituted aryl, or halo.

In certain embodiments, X₂ and X₃ are N.

In other embodiments, R₂ is H, methyl, ethyl, propyl, i-propyl, butyl,s-butyl, t-butyl, pentyl, hexyl, heptyl, phenyl, pyridinyl, pyrimidinyl,or pyrazinyl; each of which may be optionally substituted.

In a further embodiment, R₂ is substituted by 1-4 groups independentlyselected from optionally substituted alkyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aralkyl,alkoxyl, haloalkyl, halo, OH, NH₂, NHR″, CN, N₃, and NO₂, and R″ is H oralkyl.

In certain embodiments, R₁ is H, methyl, ethyl, propyl, i-propyl, butyl,i-butyl, t-butyl, pentyl, hexyl, phenyl, naphthyl, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, piperidinyl, pyrrolidinyl,tetrahydropyranyl, tetrahydrofuranyl, benzyl, phenethyl, or pyridyl,each of which may be optionally substituted.

In a further embodiment, R₁ is substituted by 1-4 groups independentlyselected from optionally substituted alkyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aralkyl,alkoxyl, haloalkoxy, haloalkyl, halo, OH, NH₂, NHR″, CN, N₃, and NO₂,and R″ is H or alkyl.

In another embodiment, R₂ is H.

In another embodiment, the invention provides a compound of formula III:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

R₂ is H, methyl, pyridyl, or phenyl, each of which may be optionallysubstituted;

R₁ is H, methyl, ethyl, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, piperidinyl, pyrrolidinyl, tetrahydropyranyl,tetrahydrofuranyl, benzyl, phenethyl, pyridyl, or phenyl, each of whichmay be optionally substituted.

In one embodiment, R₂ is optionally substituted with 1-4 groupsindependently selected from optionally substituted aryl, optionallysubstituted heteroaryl, F, Cl, Br, methyl, ethyl, propyl, methoxy, andCF₃. In certain embodiments, each of the above substituents may beoptionally substituted.

In other embodiments, R₁ is optionally substituted with 1-4 groupsindependently selected from methyl, phenyl, pyridyl, morpholino,indolyl, pyrazinyl, F, Cl, Br, methoxy, OCF₃, trifluoromethyl, OH, NH₂,CN, and NO₂. In certain embodiments, R₁ is further substituted by alkyl,aryl, haloalkyl, halo, or alkoxy. In certain embodiments, each of theabove substituents may be optionally substituted.

In another embodiment, R₂ is H.

In other embodiments, R₁ is phenyl or pyridyl, and R₂ is H or Me.

In a further embodiment, R₁ is further substituted with 1-4 substituentsindependently selected from alkyl, halo, alkoxy, haloalkyl, haloalkoxy,aryl, heteroaryl, carbocyclic, heterocyclic, and NO₂. In another furtherembodiment, R₁ is further substituted with 1-4 substituentsindependently selected from methyl, F, Cl, OCH₃, CF₃, OCF₃, phenyl,pyridyl, morpholinyl, or NO₂.

In one embodiment, the invention provides a compound of formula IV:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

R₂ is H, alkyl, haloalkyl, arylalkyl, aryl, heteroaryl, heterocyclic,carbocyclic, —C(O)R₅, or —S(O)_(p)R₅, each of which may be optionallysubstituted;

R_(x) is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted;

R_(y) is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted; or

R_(x) and R_(y) together with the carbon to which each is attached,forms a cycloalkyl or heterocycloalkyl, each of which is optionallysubstituted:

each R_(A) is independently alkyl, alkoxy, cycloalkyl, aryl,heterocycloalkyl, heteroaryl, arylalkyl, heteroarylalkyl, haloalkyl,haloalkoxy, halo, OH, —NO₂, —CN, or —NH₂; or two R_(A) groups togetherwith the atoms to which each is attached, can form an optionallysubstituted ring;

m is 0, 1, 2, 3, or 4; and

p is 0, 1, 2, or 3.

In another embodiment, the invention provides a compound of formula V:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

R₂ is H, alkyl, haloalkyl, arylalkyl, aryl, heteroaryl, heterocyclic,carbocyclic, —C(O)R₅, or —S(O)_(p)R₅, each of which may be optionallysubstituted;

R_(x) is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted;

R_(y) is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted; or

R_(x) and R_(y) together with the carbon to which each is attached,forms a cycloalkyl or heterocycloalkyl, each of which is optionallysubstituted:

each R_(A) is independently alkyl, alkoxy, cycloalkyl, aryl,heterocycloalkyl, heteroaryl, arylalkyl, heteroarylalkyl, haloalkyl,haloalkoxy, halo, OH, —NO₂, —CN, or —NH₂; or two R_(A) groups togetherwith the atoms to which each is attached, can form an optionallysubstituted ring; and

m is 0, 1, 2, 3, or 4.

In one embodiment, R₂ is H or Me.

In other embodiments, R_(x) is H, alkyl, or aryl; and R_(y) is H oralkyl; each of which is optionally substituted.

In certain embodiments, R_(x) is H, methyl, ethyl, or phenyl.

In a further embodiment, R_(y) is H, methyl or ethyl.

In other further embodiments, R_(x) and R_(y) together with the carbonto which each is attached, forms a cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, or cyclooctyl, each of which is optionallysubstituted.

In certain embodiments, R_(x) and R_(y) together with the carbon towhich each is attached, forms tetrahydropyran, piperidine, piperazine,morpholine, tetrahydrofuran, tetrahydrothiophene, pyrrolidine,oxozolidine, or imidazolidine, each of which is optionally substituted.

In still other embodiments, R_(A) is H, alkyl, alkoxy, cycloalkyl, aryl,heterocycloalkyl, heteroaryl, arylalkyl, heteroarylalkyl, haloalkyl,haloalkoxy, halo, OH, —NO₂, —CN, or —NH₂, and m is 0 or 1.

In another embodiment, the invention provides a compound of formula VI:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

R_(x) and R_(y) together with the carbon to which each is attached,forms a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, tetrahydropyran, piperidine, piperazine, morpholine,tetrahydrofuran, tetrahydrothiophene, pyrrolidine, oxozolidine, orimidazolidine, each of which is optionally substituted;

each R_(A) is independently alkyl, alkoxy, cycloalkyl, aryl,heterocycloalkyl, heteroaryl, arylalkyl, heteroarylalkyl, haloalkyl,haloalkoxy, halo, OH, —NO₂, —CN, or —NH₂; or two R_(A) groups togetherwith the atoms to which each is attached, can form an optionallysubstituted ring; and

m is 0, 1, or 2.

In one embodiment, R_(x) and R_(y) together with the carbon to whicheach is attached, forms a cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, or tetrahydropyran.

In another embodiment, m is 1 or 2, and each R_(A) is independentlymethyl, phenyl, F, Cl, methoxy, OCF₃, or CF₃; or two R_(A) groupstogether with the atoms to which each is attached, can form anoptionally substituted cycloalkyl or heterocyclic ring.

In other embodiments, m is 0.

In certain embodiments, the invention provides a compound of formula VI:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

R_(x) is H, alkyl, or aryl; each of which is optionally substituted;

R_(y) is H or alkyl; each of which is optionally substituted;

each R_(A) is independently alkyl, alkoxy, cycloalkyl, aryl,heterocycloalkyl, heteroaryl, arylalkyl, heteroarylalkyl, haloalkyl,haloalkoxy, halo, OH, —NO₂, —CN, or —NH₂; or two R_(A) groups togethercan form an optionally substituted cycloalkyl or heterocyclic ring; and

m is 0, 1, or 2.

In some embodiments, R_(x) is H, methyl, ethyl, or phenyl.

In other embodiments, R_(y) is H, methyl or ethyl.

In certain embodiments, m is 1 or 2, and each R_(A) is independentlymethyl, phenyl, F, Cl, methoxyl, or CF₃; or two R_(A) groups togethercan form an optionally substituted cycloalkyl or heterocyclic ring.

In other embodiments, m is 0.

In another embodiment, the invention provides a compound of formula VII:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

ring A is a heteroaryl or heterocyclic;

R₂ is H, alkyl, haloalkyl, arylalkyl, aryl, heteroaryl, heterocyclic,carbocyclic, —C(O)R₅, or —S(O)_(p)R₅, each of which may be optionallysubstituted;

R_(x) is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted;

R_(y) is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted; or

R_(x) and R_(y) together with the carbon to which each is attached,forms a cycloalkyl or heterocycloalkyl, each of which is optionallysubstituted:

each R_(A) is independently alkyl, alkoxy, cycloalkyl, aryl,heterocycloalkyl, heteroaryl, arylalkyl, heteroarylalkyl, haloalkyl,haloalkoxy, halo, OH, —NO₂. —CN, or —NH₂; or two R_(A) groups togethercan form an optionally substituted cycloalkyl or heterocyclic ring;

m is 0, 1, 2, 3, or 4; and

p is 0, 1, 2, or 3.

In another embodiment, the invention provides a compound of formulaVII-A:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

ring A is a heteroaryl or heterocyclic;

R₂ is H, alkyl, haloalkyl, arylalkyl, aryl, heteroaryl, heterocyclic,carbocyclic, —C(O)R₅, or —S(O)_(p)R₅, each of which may be optionallysubstituted;

R_(x) is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted;

R_(y) is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted; or

R_(x) and R_(y) together with the carbon to which each is attached,forms a cycloalkyl or heterocycloalkyl, each of which is optionallysubstituted:

each R_(A) is independently alkyl, alkoxy, cycloalkyl, aryl,heterocycloalkyl, heteroaryl, arylalkyl, heteroarylalkyl, haloalkyl,haloalkoxy, halo, OH, —NO₂, —CN, or —NH₂; or two R_(A) groups togethercan form an optionally substituted cycloalkyl or heterocyclic ring; and

m is 0, 1, 2, 3, or 4.

In certain embodiments, ring A is acridinyl, azocinyl, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,benzisothiazolyl, benzimidazolinyl, carbazolyl, NH-carbazolyl,carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl,furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl,indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl; 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, pteridinyl, purinyl, pyranyl, pyrazinyl,pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,2,5-thiadiazolyl, 1,3,4thiadiazolyl, thianthrenyl, thiazolyl, thienyl,thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl,triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl,1,3,4-triazolyl, or xanthenyl.

In other embodiments, R₂ is H or Me.

In various embodiments, R_(x) is H, alkyl, aryl, or heteroaryl.

In certain embodiments, R_(y) is H, alkyl, aryl, or heteroaryl.

In various embodiments, R_(y) is H, methyl or pyridine, and R_(y) is H.

In certain embodiments, R_(x) and R_(y) together with the carbon towhich each is attached, forms a cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, or cyclooctyl, each of which is optionallysubstituted.

In another embodiment, R_(x) and R_(y) together with the carbon to whicheach is attached, forms tetrahydropyran, piperidine, piperazine,morpholine, tetrahydrofuran, tetrahydrothiophene, pyrrolidine,oxozolidine, or imidazolidine, each of which is optionally substituted.

In various embodiments, R_(A) is H, alkyl, alkoxy, cycloalkyl, aryl,heterocycloalkyl, heteroaryl, arylalkyl, heteroarylalkyl, haloalkyl,haloalkoxy, halo, OH, —NO₂, —CN, or —NH₂, and m is 0 or 1.

In another embodiment, the invention provides a compound of formulaVIII:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

ring A is benzofuranyl, benzothiofuranyl, furanyl, imidazolinyl,imidazolyl, isoquinolinyl, morpholinyl, oxazolidinyl, pyrimidinyl,piperazinyl, piperidinyl, pyridinyl, pyridyl, pyrazinyl, pyrimidinyl,pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,quinolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, thiophenyl, or triazinyl;

R_(x) and R_(y) together with the carbon to which each is attached,forms a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, tetrahydropyran, piperidine, piperazine, morpholine,tetrahydrofuran, tetrahydrothiophene, pyrrolidine, oxozolidine, orimidazolidine, each of which is optionally substituted;

each R_(A) is independently alkyl, alkoxy, cycloalkyl, aryl,heterocycloalkyl, heteroaryl, arylalkyl, heteroarylalkyl, haloalkyl,haloalkoxy, halo, OH, —NO₂, —CN, or —NH₂; or two R_(A) groups togethercan form an optionally substituted cycloalkyl or heterocyclic ring; and

m is 0, 1, or 2.

In certain embodiments, ring A is benzofuranyl, benzothiofuranyl,furanyl, pyridyl, pyrazinyl, pyrimidinyl, or thiophenyl.

In other embodiments, R_(x) and R_(y) together with the carbon to whicheach is attached, forms a cyclopropyl, cyclopentyl, cyclohexyl, ortetrahydropyran.

In certain embodiments, m is 1, and R_(A) is F, Cl, methoxyl, or CF₃.

In other embodiments, m is 0.

Representative compounds of the invention include, but are not limitedto, the following compounds of Table 1 below.

TABLE 1

   1

   2

   3

   4

   5

   6

   7

   8

   9

   10

   11

   12

   13

   14

   15

   16

   17

   18

   19

   20

   21

   22

   23

   24

   25

   26

   27

   28

   29

   30

   31

   32

   33

   34

   35

   36

   37

   38

   39

   40

   41

   42

   43

   44

   45

   46

   47

   48

   49

   50

   51

   52

   53

   54

   55

   57

   58

   59

   60

   61

   62

   63

   65

   66

   67

   68

   69

   70

   71

   72

   73

   74

   75

   76

   77

   78

   79

   80

   81

   82

   83

   84

   85

   86

   97

   87

   88

   89

   90

   91

   92

   93

   94

   95

   96

   98

   99

  100

  101

  102

  103

  104

  105

  106

  107

  108

  109

  110

  111

  112

  113

  114

  115

  116

  117

  118

  119

  120

  121

  122

  123

  124

  125

  126

  127

  128

  129

  130

  131

  132

  133

  134

  135

  136

  137

  138

  139

  140

  141

  142

  143

  144

  145

  146

  147

  148

  149

  150

  151

  152

  153

  154

  155

In certain embodiments, the compound of the invention is selected fromTable 2;

TABLE 2

  73

  81

  82

  84

  97

  87

  88

  90

  91

  93

  94

  95

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound of any of the formulae herein, (e.g., formula I):

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

each of X₁, X₂, X₃, or X₄ is independently N or CR′, wherein 2 of X₁,X₂, X₃, or X₄ are N;

each R′ is independently H, optionally substituted alkyl, optionallysubstituted aryl, or halo;

R₁ is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted; and

R₂ is H, alkyl, haloalkyl, alkenyl, arylalkyl, aryl, heteroaryl,heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅, each of which may beoptionally substituted;

p is 0, 1, or 2;

each R₅ is independently H; optionally substituted alkyl, optionallysubstituted carbocyclic, optionally substituted heterocyclic, optionallysubstituted aryl, or optionally substituted heteroaryl;

wherein R₁ and R₂ are both not simultaneously H; together with apharmaceutically acceptable carrier.

In preferred embodiments, a compound useful in the invention has one ormore of the following properties: the compound is capable of inhibitingat least one histone deacetylase; the compound is capable of inhibitingHDAC6: the compound is a selective HDAC6 inhibitor: the compound bindsto the poly-ubiquitin binding domain of HDAC6; the compound is capableof inducing apoptosis in cancer cells (especially multiple myelomacells, non-Hodgkin's lymphoma (NML) cells, breast cancer cells, acutemyelogeous leukemia (AML) cells); and/or the compound is capable ofinhibiting aggresome formation.

In certain preferred embodiments, a compound of the invention comprisesa metal binding moiety, preferably a zinc-binding moiety such as ahydroxamate. As noted above, certain hydroxamates are potent inhibitorsof HDAC6 activity; without wishing to be bound by theory, it is believedthat the potency of these hydroxamates is due, at least in part, to theability of the compounds to bind zinc. In preferred embodiments, acompound of the invention includes at least one portion or region whichcan confer selectivity for a biological target implicated in theaggresome pathway, e.g., a biological target having tubulin deacetylase(TDAC) or HDAC activity, e.g., HDAC6. Thus, in certain preferredembodiments, a compound of the invention includes a zinc-binding moietyspaced from other portions of the molecule which are responsible forbinding to the biological target.

The invention also provides for a pharmaceutical composition comprisinga compound of any of the formulae herein (e.g., formula I), or apharmaceutically acceptable ester, salt, or prodrug thereof, togetherwith a pharmaceutically acceptable carrier.

Another object of the present invention is the use of a compound asdescribed herein (e.g., of any formulae herein) in the manufacture of amedicament for use in the treatment of a disorder or disease herein.Another object of the present invention is the use of a compound asdescribed herein (e.g., of any formulae herein) for use in the treatmentof a disorder or disease herein.

In another aspect, the invention provides a method of synthesizing acompound of any of the formulae herein (e.g., formula I). The synthesisof the compounds of the invention can be found in the Examples below.

Another embodiment is a method of making a compound of any of theformulae herein using any one, or combination of, reactions delineatedherein. The method can include the use of one or more intermediates orchemical reagents delineated herein.

Another aspect is an isotopically labeled compound of any of theformulae delineated herein. Such compounds have one or more isotopeatoms which may or may not be radioactive (e.g., ¹H, ²H, ¹⁴C, ¹³C, ³⁵S,³²P, ¹²⁵I, and ¹³¹I) introduced into the compound. Such compounds areuseful for drug metabolism studies and diagnostics, as well astherapeutic applications.

A compound of the invention can be prepared as a pharmaceuticallyacceptable acid addition salt by reacting the free base form of thecompound with a pharmaceutically acceptable inorganic or organic acid.Alternatively, a pharmaceutically acceptable base addition salt of acompound of the invention can be prepared by reacting the free acid formof the compound with a pharmaceutically acceptable inorganic or organicbase.

Alternatively, the salt forms of the compounds of the invention can beprepared using salts of the starting materials or intermediates.

The free acid or free base forms of the compounds of the invention canbe prepared from the corresponding base addition salt or acid additionsalt from, respectively. For example a compound of the invention in anacid addition salt form can be converted to the corresponding free baseby treating with a suitable base (e.g., ammonium hydroxide solution,sodium hydroxide, and the like). A compound of the invention in a baseaddition salt form can be converted to the corresponding free acid bytreating with a suitable acid (e.g., hydrochloric acid, etc.).

Prodrug derivatives of the compounds of the invention can be prepared bymethods known to those of ordinary skill in the art (e.g., for furtherdetails see Saulnier et al., (1994), Bioorganic and Medicinal ChemistryLetters, Vol. 4, p. 1985). For example, appropriate prodrugs can beprepared by reacting a non-derivatized compound of the invention with asuitable carbamylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate,para-nitrophenyl carbonate, or the like).

Protected derivatives of the compounds of the invention can be made bymeans known to those of ordinary skill in the art. A detaileddescription of techniques applicable to the creation of protectinggroups and their removal can be found in T. W. Greene, “ProtectingGroups in Organic Chemistry”, 3rd edition, John Wiley and Sons, Inc.,1999, and subsequent editions thereof.

Compounds of the present invention can be conveniently prepared, orformed during the process of the invention, as solvates (e.g.,hydrates). Hydrates of compounds of the present invention can beconveniently prepared by recrystallization from an aqueous/organicsolvent mixture, using organic solvents such as dioxan, tetrahydrofuranor methanol.

Acids and bases useful in the methods herein are known in the art. Acidcatalysts are any acidic chemical, which can be inorganic (e.g.,hydrochloric, sulfuric, nitric acids, aluminum trichloride) or organic(e.g., camphorsulfonic acid, p-toluenesulfonic acid, acetic acid,ytterbium triflate) in nature. Acids are useful in either catalytic orstoichiometric amounts to facilitate chemical reactions. Bases are anybasic chemical, which can be inorganic (e.g., sodium bicarbonate,potassium hydroxide) or organic (e.g., triethylamine, pyridine) innature. Bases are useful in either catalytic or stoichiometric amountsto facilitate chemical reactions.

In addition, some of the compounds of this invention have one or moredouble bonds, or one or more asymmetric centers. Such compounds canoccur as racemates, racemic mixtures, single enantiomers, individualdiastereomers, diastereomeric mixtures, and cis- or trans- or E- orZ-double isomeric forms, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)-, or as(D)- or (L)- for amino acids. All such isomeric forms of these compoundsare expressly included in the present invention. Optical isomers may beprepared from their respective optically active precursors by theprocedures described above, or by resolving the racemic mixtures. Theresolution can be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques which are known to those skilled in the art. Furtherdetails regarding resolutions can be found in Jacques, et al.,Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). Thecompounds of this invention may also be represented in multipletautomeric forms, in such instances, the invention expressly includesall tautomeric forms of the compounds described herein. When thecompounds described herein contain olefinic double bonds or othercenters of geometric asymmetry, and unless specified otherwise, it isintended that the compounds include both E and Z geometric isomers.Likewise, all tautomeric forms are also intended to be included. Theconfiguration of any carbon-carbon double bond appearing herein isselected for convenience only and is not intended to designate aparticular configuration unless the text so states; thus a carbon-carbondouble bond depicted arbitrarily herein as trans may be cis, trans, or amixture of the two in any proportion. All such isomeric forms of suchcompounds are expressly included in the present invention. All crystalforms of the compounds described herein are expressly included in thepresent invention.

The synthesized compounds can be separated from a reaction mixture andfurther purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. In addition, the solvents, temperatures, reaction durations,etc. delineated herein are for purposes of illustration only and one ofordinary skill in the art will recognize that variation of the reactionconditions can produce the desired compounds of the present invention.Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing the compoundsdescribed herein are known in the art and include, for example, thosesuch as described in R. Larock, Comprehensive Organic Transformations,VCH Publishers (1989): T. W. Greene and P. G. M. Wuts, Protective Groupsin Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser andM. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, JohnWiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagentsfor Organic Synthesis, John Wiley and Sons (1995), and subsequenteditions thereof.

In embodiments, the invention provides for the intermediate compounds ofthe formulae delineated herein and methods of converting such compoundsto compounds of the formulae herein (e.g., in schemes herein) comprisingreacting a compound herein with one or more reagents in one or morechemical transformations (including those provided herein) to therebyprovide the compound of any of the formulae herein or an intermediatecompound thereof.

The synthetic methods described herein may also additionally includesteps, either before or after any of the steps described in any scheme,to add or remove suitable protecting groups in order to ultimately allowsynthesis of the compound of the formulae described herein. The methodsdelineated herein contemplate converting compounds of one formula tocompounds of another formula (e.g., in Scheme A, A1 to A2; A2 to A3; A1to A3). The process of converting refers to one or more chemicaltransformations, which can be performed in situ, or with isolation ofintermediate compounds. The transformations can include reacting thestarting compounds or intermediates with additional reagents usingtechniques and protocols known in the art, including those in thereferences cited herein. Intermediates can be used with or withoutpurification (e.g., filtration, distillation, sublimation,crystallization, trituration, solid phase extraction, andchromatography).

The compounds of this invention may be modified by appending variousfunctionalities via any synthetic means delineated herein to enhanceselective biological properties. Such modifications are known in the artand include those which increase biological penetration into a givenbiological system (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

The compounds of the invention are defined herein by their chemicalstructures and/or chemical names. Where a compound is referred to byboth a chemical structure and a chemical name, and the chemicalstructure and chemical name conflict, the chemical structure isdeterminative of the compound's identity.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentherein includes that embodiment as any single embodiment or incombination with any other embodiments or portions thereof. Therecitation of an embodiment for a variable herein includes thatembodiment as any single embodiment or in combination with any otherembodiments or portions thereof.

METHODS OF THE INVENTION

In another aspect, the invention provides a method of selectivelyinhibiting HDAC6 over other HDACs (e.g. HDAC1, HDAC2, HDAC3) in asubject, comprising administering to the subject a compound of formulaI:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

each of X₁, X₂, X₃, or X₄ is independently N or CR′, wherein 2 of X₁,X₂, X₃, or X₄ are N;

each R′ is independently H, optionally substituted alkyl, optionallysubstituted aryl, or halo;

R₁ is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted; and

R₂ is H, alkyl, haloalkyl, alkenyl, arylalkyl, aryl, heteroaryl,heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅, each of which may beoptionally substituted:

p is 0, 1, or 2;

each R₅ is independently H; optionally substituted alkyl, optionallysubstituted carbocyclic, optionally substituted heterocyclic, optionallysubstituted aryl, or optionally substituted heteroaryl;

wherein R₁ and R₂ are both not simultaneously H.

In one embodiment, the compound of any of the formulae herein (e.g.,formula I) has a selectivity for HDAC6 of 5 to 1000 fold over otherHDACs.

In another embodiment, the compound of of any of the formulae herein(e.g., formula I) has a selectivity for HDAC6 when tested in a HDACenzyme assay of about 5 to 1000 fold over other HDACs.

In certain embodiments, the compound has a selectivity for HDAC6 of 15to 40 fold over other HDACs.

In another aspect, the invention provides a method of treating a diseasemediated by HDAC6 in a subject comprising administering to the subject acompound of formula I:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

each of X₁, X₂, X₃, or X₄ is independently N or CR′, wherein 2 of X₁,X₂, X₃, or X₄ are N;

each R′ is independently H, optionally substituted alkyl, optionallysubstituted aryl, or halo;

R₁ is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted; and

R₂ is H, alkyl, haloalkyl, alkenyl, arylalkyl, aryl, heteroaryl,heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅, each of which may beoptionally substituted:

p is 0, 1, or 2;

each R₅ is independently H; optionally substituted alkyl, optionallysubstituted carbocyclic, optionally substituted heterocyclic, optionallysubstituted aryl, or optionally substituted heteroaryl;

wherein R₁ and R₂ are both not simultaneously H.

In certain embodiments, the disease is cancer or a proliferationdisease.

In a further embodiment, the disease is lung cancer, colon cancer,breast cancer, prostate cancer, liver cancer, brain cancer, kidneycancer, ovarian cancer, stomach cancer, skin cancer, bone cancer,gastric cancer, pancreatic cancer, glioma, gliobastoma, hepatocellularcarcinoma, papillary renal carcinoma, head and neck squamous cellcarcinoma, leukemias, lymphomas, myelomas, and solid tumors.

In a further embodiment, the cancer is multiple myeloma.

In other embodiments, the disease is Wilson's disease, spinocerebellarataxia, prion disease, Parkinson's disease, Huntington's disease,amytrophic lateral sclerosis, amyloidosis, Alzheimer's disease,Alexander's disease, alcoholic liver disease, cystic fibrosis, Pick'sDisease, spinal muscular dystrophy or Lewy body dementia.

In other embodiments, the disease is an inflammatory, immune andautoimmune diseases including, but not limited to, arthritic conditions,such as, rheumatoid arthritis, osteoarthritis; juvenile arthritis,rheumatoid spondylitis; psoriasis; post ischemic perfusion injury;inflammatory bowel disease; chronic inflammatory pulmonary disease,eczema, asthma, psoriasis, ischemia/reperfusion injury, ulcerativecolitis, acute respiratory distress syndrome, psoriatic arthritis,infectious arthritis, progressive chronic arthritis, deformingarthritis, osteoarthritis, traumatic arthritis, gouty arthritis,Reiter's syndrome, polychondritis, acute synovitis and spondylitis,glomerulonephritis (with or without nephrotic syndrome), autoimmunehematologic disorders (e.g. hemolytic anemia, aplastic anemia,idiopathic thrombocytopenia and neutropenia) ulcerative colitis, Crohn'sdisease, host versus graft disease, graft versus host disease, allograftrejection, chronic thyroiditis, Graves' disease, schleroderma, diabetes(type I and type II), active hepatitis (acute and chronic), primarybinary cirrhosis, myasthenia gravis, multiple sclerosis (MS), systemiclupus erythematosus, atopic dermatitis, contact dermatitis, skinsunburns, chronic renal insufficiency, Stevens-Johnson syndrome,idiopathic sprue, sarcoidosis, Guillain-Barre syndrome, uveitis,conjunctivitis, keratoconjunctivitis, otitis media, periodontal disease,pulmonary interstitial fibrosis, asthma, bronchitis, rhinitis,sinusitis, pneumoconiosis, pulmonary insufficiency syndrome, pulmonaryemphysema, pulmonary fibrosis, silicosis, chronic inflammatory pulmonarydisease (e.g. chronic obstructive pulmonary disease) and otherinflammatory or obstructive diseases of the airways.

In one embodiment, the HDAC6 inhibitors of the invention are useful fortreating any one or more of the following autoimmune diseases ordisorders, systemic lupus erythematosis, autoimmune thyroiditis,dermatitis (including atopic dermatitis and eczematous dermatitis),psoriasis, including keratoconjunctivitis sicca secondary to Sjögren'sSyndrome, alopecia areata, allergic responses due to arthropod bitereactions, aphthous ulcer, iritis, conjunctivitis, allergic asthma,cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drugeruptions, leprosy reversal reactions, erythema nodosum leprosum,autoimmune uveitis, allergic encephalomyelitis, acute necrotizinghemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, idiopathic thrombocytopenia, polychondritis,Wegener's granulomatosis, chronic active hepatitis, lichen planus,sarcoidosis, primary biliary cirrhosis, uveitis posterior, andinterstitial lung fibrosis.

Additionally, the methods of the invention may also be useful in thetreatment of protozoal infections. The methods of the invention are alsouseful in the treatment of diseases associated with aberrant proteincatabolism, for example, protein degradation disorders, disordersassociated with misfolded proteins, and protein deposition disorders. Incertain embodiments, the HDAC6 inhibitors of the invention are useful inthe treatment of the protein deposition disorders, Wilson's disease,spinocerebellar ataxia, prion disease, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, spinal muscular atrophy, spinaland bulbar muscular atrophy, amyloidosis, Alzheimer's disease,Alexander's disease, alcoholic liver disease, cystic fibrosis, Pick'sdisease, and Lewy body dementia. In certain exemplary embodiments, theclaimed methods of the invention are useful for treating disordersassociated with histone deacetylation activity. In certain exemplaryembodiments, the methods of the invention are useful for treatingdisorders associated with tubulin deacetylation activity.

Neurodegenerative diseases that can be treated or prevented includeAlzheimer's disease, Parkinson's disease, cerebral ischaemia, traumaticneurodegenerative disease, Huntington's disease or chorea, seniledementia, memory disorder, vascular dementia, lesions associated withcerebral ischemia (stroke) and with cranial and medullary trauma, amongothers.

Methods delineated herein include those wherein the subject isidentified as in need of a particular stated treatment. Identifying asubject in need of such treatment can be in the judgment of a subject ora health care professional and can be subjective (e.g. opinion) orobjective (e.g. measurable by a test or diagnostic method).

Preferably, the HDAC6 inhibitors are selective inhibitors of HDAC6 and,as such, are useful in the treatment of disorders modulated by histonedeacetylases. In one embodiment, the HDAC6 inhibitors of the inventionare selective inhibitors of tubulin deacetylases and, as such, areuseful in the treatment of disorders modulated by tubulin deacetylases.

Thus, in another aspect of the invention, methods for the treatment ofcancer are provided comprising administering a therapeutically effectiveamount of an HDAC6 inhibitor, as described herein, to a subject in needthereof. In certain embodiments, the subject is identified as in need ofsuch treatment. In certain embodiments, a method for the treatment of adiseases is provided comprising administering a therapeuticallyeffective amount of an HDAC6 inhibitor, or a pharmaceutical compositioncomprising an HDAC6 inhibitor to a subject in need thereof, in suchamounts and for such time as is necessary to achieve the desired result.In certain embodiments of the present invention a “therapeuticallyeffective amount” of an HDAC6 inhibitor or pharmaceutical composition isthat amount effective for modulating a subset of a group of cytokinesaccording to the invention.

In certain embodiments, the method involves the administration of atherapeutically effective amount of an HDAC6 inhibitor or apharmaceutically acceptable derivative thereof to a subject (including,but not limited to a human or animal) in need of it (including a subjectidentified as in need). In certain embodiments, the HDAC6 inhibitors areuseful for the treatment of cancer (including, but not limited to,glioblastoma, retinoblastoma, breast cancer, cervical cancer, colon andrectal cancer, leukemia (e.g., CML, AML, CLL, ALL), lymphoma, lungcancer (including, but not limited to small cell lung cancer), melanomaand/or skin cancer, multiple myeloma, non-Hodgkin's lymphoma, ovariancancer, pancreatic cancer, prostate cancer and gastric cancer, bladdercancer, uterine cancer, kidney cancer, testicular cancer, stomachcancer, brain cancer, liver cancer, or esophageal cancer, melanoma ormultiple melanoma). In certain embodiments, the HDAC6 inhibitors of theinvention are active against leukemia cells and melanoma cells, and thusare useful for the treatment of leukemias (e.g., myeloid, lymphocytic,myelocytic and lymphoblastic leukemias) and malignant melanomas. Instill other embodiments, the inventive anticancer agents are activeagainst solid tumors. Accordingly, in yet another aspect, according tothe methods of treatment of the present invention, tumor cells arekilled, or their growth is inhibited by contacting said tumor cells withan HDAC6 inhibitor, as described herein.

In certain embodiments, the invention provides a method of treatment ofany of the disorders described herein, wherein the subject is a human.

In accordance with the foregoing, the present invention further providesa method for preventing or treating any of the diseases or disordersdescribed above in a subject in need of such treatment, which methodcomprises administering to said subject a therapeutically effectiveamount of an HDAC6 inhibitor of the invention or a pharmaceuticallyacceptable salt thereof. For any of the above uses, the required dosagewill vary depending on the mode of administration, the particularcondition to be treated and the effect desired.

In another aspect, the invention provides a method of treating a subjectsuffering from or susceptible to multiple myeloma comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound of formula I:

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

each of X₁, X₂, X₃, or X₄ is independently N or CR′, wherein 2 of X₁,X₂, X₃, or X₄ are N;

each R′ is independently H, optionally substituted alkyl, optionallysubstituted aryl, or halo;

R₁ is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted; and

R₂ is H, alkyl, haloalkyl, alkenyl, arylalkyl, aryl, heteroaryl,heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅, each of which may beoptionally substituted:

p is 0, 1, or 2;

each R₅ is independently H: optionally substituted alkyl, optionallysubstituted carbocyclic, optionally substituted heterocyclic, optionallysubstituted aryl, or optionally substituted heteroaryl;

wherein R₁ and R₂ are both not simultaneously H;

to thereby treat the subject suffering from or susceptible to multiplemyeloma.

In another embodiment, the methods herein comprise a step wherein acompound is identified as having a Z-fold selectivity for HDAC6 overother HDACs (e.g., HDAC1, HDAC2, HDAC3), wherein Z is an integer between2 and 1000.

In one embodiment, the invention provides a method further comprisingco-administering one or more of a chemotherapeutic agent, radiationagent, hormonal agent, biological agent or an anti-inflammatory agent tothe subject.

In certain embodiments, the chemotherapeutic agent is tamoxifen,trastuzamab, raloxifene, doxorubicin, fluorouracil/5-fu, pamidronatedisodium, anastrozole, exemestane, cyclophosphamide, epirubicin,letrozole, toremifene, fulvestrant, fluoxymesterone, trastuzumab,methotrexate, megastrol acetate, docetaxel, paclitaxel, testolactone,aziridine, vinblastine, capecitabine, goselerin acetate, zoledronicacid, taxol, vinblastine, or vincristine.

In other embodiments, the invention provides any method as describedherein, wherein the subject is a human.

In another aspect, the invention provides a kit comprising a compoundcapable of inhibiting HDAC activity selected from one or more compoundsof any of the formulae herein (e.g., formula I),

or a pharmaceutically acceptable salt, ester or prodrug thereof,

wherein,

each of X₁, X₂, X₃, or X₄ is independently N or CR′, wherein 2 of X₁,X₂, X₃, or X₄ are N;

each R′ is independently H, optionally substituted alkyl, optionallysubstituted aryl, or halo;

R₁ is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted; and

R₂ is H, alkyl, haloalkyl, alkenyl, arylalkyl, aryl, heteroaryl,heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅, each of which may beoptionally substituted;

p is 0, 1, or 2;

each R₅ is independently H; optionally substituted alkyl, optionallysubstituted carbocyclic, optionally substituted heterocyclic, optionallysubstituted aryl, or optionally substituted heteroaryl;

wherein R₁ and R₂ are both not simultaneously H;

and instructions for use in treating multiple myeloma.

As discussed above, the present invention provides compounds useful forthe treatment of various diseases. In certain embodiments, the compoundsof the present invention are useful as inhibitors of histone or tubulindeacetylases and thus are useful as anti-cancer agents, and thus may beuseful in the treatment of cancer, by effecting tumor cell death orinhibiting the growth of tumor cells. In certain exemplary embodiments,the inventive anticancer agents are useful in the treatment of cancersand other proliferative disorders, as described above. In certainembodiments, the inventive anticancer agents are active against leukemiacells and melanoma cells, and thus are useful for the treatment ofleukemias (e.g., myeloid, lymphocytic, myelocytic and lymphoblasticleukemias) and malignant melanomas. In certain embodiments, thecompounds are useful in the treatment of multiple myeloma.

The compounds of the invention are also effective to treat or preventautoimmune hematologic disorders (e.g. hemolytic anemia, aplasic anemia,idiopathic thrombocytopenia and neutropenia), chronic inflammatorypulmonary disease (e.g. chronic obstructive pulmonary disease) and otherinflammatory or obstructive diseases of the airways.

Additionally, the inventive compounds may also be useful in thetreatment of protozoal infections. The inventive compounds are alsouseful in the treatment of diseases associated with aberrant proteincatabolism, for example, protein degradation disorders, disordersassociated with misfolded proteins, and protein deposition disorders. Incertain embodiments, the compounds are useful in the treatment of theprotein deposition disorders, Wilson's disease, spinocerebellar ataxia,prion disease, Parkinson's disease, Huntington's disease, amyotrophiclateral sclerosis, spinal muscular atrophy, spinal and bulbar muscularatrophy, amyloidosis, Alzheimer's disease, Alexander's disease,alcoholic liver disease, cystic fibrosis, Pick's disease, and Lewy bodydementia. In certain exemplary embodiments, the compounds of theinvention are useful for disorders associated with histone deacetylationactivity. In certain exemplary embodiments, the compounds of theinvention are useful for disorders associated with tubulin deacetylationactivity.

Neurodegenerative diseases that can be treated or prevented includeAlzheimer's disease, Parkinson's disease, cerebral ischaemia, traumaticneurodegenerative disease, Huntingtons's disease or chorea, seniledementia, memory disorder, vascular dementia, lesions associated withcerebral ischemia (stroke) and with cranial and medullary trauma, amongothers.

Methods delineated herein include those wherein the subject isidentified as in need of a particular stated treatment. Identifying asubject in need of such treatment can be in the judgment of a subject ora health care professional and can be subjective (e.g. opinion) orobjective (e.g. measurable by a test or diagnostic method).

As discussed above, the compounds of the invention are selectiveinhibitors of HDAC6 and, as such, are useful in the treatment ofdisorders modulated by histone deacetylases. As discussed above, thecompounds of the invention are selective inhibitors of tubulindeacetylases and, as such, are useful in the treatment of disordersmodulated by tubulin deacetylases. For example, compounds of theinvention may be useful in the treatment of cancer (e.g., breast cancer,prostate cancer, multiple myeloma, leukemia, lymphoma, etc.).Accordingly, in yet another aspect, according to the methods oftreatment of the present invention, tumor cells are killed, or theirgrowth is inhibited by contacting said tumor cells with an inventivecompound or composition, as described herein.

Thus, in another aspect of the invention, methods for the treatment ofcancer are provided comprising administering a therapeutically effectiveamount of an inventive compound (i.e., of any of the formulae herein),as described herein, to a subject in need thereof. In certainembodiments, the subject is identified as in need of such treatment. Incertain embodiments, a method for the treatment of cancer is providedcomprising administering a therapeutically effective amount of aninventive compound, or a pharmaceutical composition comprising aninventive compound to a subject in need thereof, in such amounts and forsuch time as is necessary to achieve the desired result. In certainembodiments of the present invention a “therapeutically effectiveamount” of the inventive compound or pharmaceutical composition is thatamount effective for killing or inhibiting the growth of tumor cells.The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for killing or inhibiting the growth of tumorcells. Thus, the expression “amount effective to kill or inhibit thegrowth of tumor cells,” as used herein, refers to a sufficient amount ofagent to kill or inhibit the growth of tumor cells. The exact amountrequired will vary from subject to subject, depending on the species,age, and general condition of the subject, the severity of theinfection, the particular anticancer agent, its mode of administration,and the like.

In certain embodiments, the method involves the administration of atherapeutically effective amount of the compound or a pharmaceuticallyacceptable derivative thereof to a subject (including, but not limitedto a human or animal) in need of it.

Multiple myeloma (MM) is a plasma cell malignancy which remainsincurable despite conventional treatment (Gregory, et al. (1992) J ClinOncol 10, 334-342) as well as high dose therapy and stem celltransplantation (Attal, M., et al. (2003) N Engl J Med 349, 2495-2502).Novel agents have recently been developed which target not only MMcells, but also the bone marrow (BM) microenvironment, and can overcomeconventional drug resistance (Hideshima, T. & Anderson, K. C. (2002) NatRev Cancer 2, 927-937). For example, the proteasome inhibitor bortezomib(formally PS-341) induces significant anti-tumor activity in human MMcell lines and freshly isolated patient MM cells (Hideshima, T. &Anderson, K. C. (2002) Nat Rev Cancer 2, 927-937; Hideshima, et al.(2001) Cancer Res. 61, 3071-3076; Mitsiades, N., et al. (2002) Proc NatlAcad Sci USA 99, 14374-14379; Hideshima, T., et al. (2002) J Biol Chem277, 16639-47: Mitsiades, N., et al. (2003) Blood 101, 2377-80; Chauhan,D., et al (2003) Cancer Res 63, 6174-6177; Hideshima, T., et al. (2003)Blood 101, 1530-1534; Hideshima, T., et al. (2003) Oncogene 22,8386-8393; Hideshima, T., et al. (2004) Oncogene 23, 8766-8776)associated with c-Jun NH2-terminal kinase (JNK) (also known asstress-activated protein kinase) and caspase activation, followed byapoptosis (Hideshima, T., et al. (2001) Cancer Res. 61, 3071-3076;Mitsiades, N., et al. (2002) Proc Natl Acad Sci USA 99, 14374-14379;Hideshima, T., et al. (2003) Blood 101, 1530-1534). Bortezomib alsoinhibits adherence of MM cells to bone marrow stromal cells (BMSCs) bydownregulating adhesion molecules (ICAM-I and VCAM-I) (Hideshima, T., etal. (2001) Oncogene 20, 4519-4527); as well as induces cleavage ofDNA-protein kinase catalytic subunit and ataxia telangiectasia mutated,suggesting that bortezomib also inhibits DNA repair. Neither IL-6 noradherence of MM cells to BMSCs protects against bortezomib-inducedapoptosis. Without wishing to be bound by any scientific theory,bortezomib enhances sensitivity and can overcome resistance in MM cellsto conventional chemotherapeutic agents, especially to DNA damagingagents (Mitsiades, N., et al. (2003) Blood 101, 2377-80). In support ofthis, a phase II trial of bortezomib treatment of 202 patients withrefractory relapsed MM demonstrated 35% responses, including 10%complete and near complete responses (Richardson, P. G., et al. (2003)NEnglJMed 348, 2609-2617); however, 65% of patients did not respond.Heat-shock protein (hsp)-27 mediates bortezomib-resistance; conversely,inhibiting hsp-27 expression using hsp-27 antisense, p38mitogen-activated protein kinase (MAPK) siRNA, or p38 MAPK inhibitor todownregulate hsp-27 can restore MM cell susceptibility to bortezomib(Chauhan, D., et al. (2003) Cancer Res 63, 6174-6177; Hideshima, T., etal. (2004) Oncogene 23, 8766-8776).

In certain embodiments, the inventive compounds also find use in theprevention of restenosis of blood vessels subject to traumas such asangioplasty and stenting. For example, it is contemplated that thecompounds of the invention will be useful as a coating for implantedmedical devices, such as tubings, shunts, catheters, artificialimplants, pins, electrical implants such as pacemakers, and especiallyfor arterial or venous stents, including balloon-expandable stents. Incertain embodiments inventive compounds may be bound to an implantablemedical device, or alternatively, may be passively adsorbed to thesurface of the implantable device. In certain other embodiments, theinventive compounds may be formulated to be contained within, or,adapted to release by a surgical or medical device or implant, such as,for example, stents, sutures, indwelling catheters, prosthesis, and thelike. Accordingly, without wishing to be bound to any particular theory,the inventive compounds having antiproliferative effects can be used asstent coatings and/or in stent drug delivery devices, inter alia for theprevention of restenosis or reduction of restenosis rate. Suitablecoatings and the general preparation of coated implantable devices aredescribed in U.S. Pat. Nos. 6,099,562; 5,886,026; and 5,304,121; each ofwhich is incorporated herein by reference. The coatings are typicallybiocompatible polymeric materials such as a hydrogel polymer,polymethyldisiloxane. polycaprolactone, polyethylene glycol, polylacticacid, ethylene vinyl acetate, and mixtures thereof. The coatings mayoptionally be further covered by a suitable topcoat of fluorosilicone,polysaccarides, polyethylene glycol, phospholipids or combinationsthereof to impart controlled release characteristics in the composition.A variety of compositions and methods related to stent coating and/orlocal stent drug delivery for preventing restenosis are known in the art(see, for example, U.S. Pat. Nos. 6,517,889; 6,273,913; 6,258,121;6,251,136; 6,248,127; 6,231,600; 6,203,551; 6,153,252; 6,071,305;5,891,507; 5,837,313 and published U.S. patent application No.:US2001/0027340, each of which is incorporated herein by reference in itsentirety). For example, stents may be coated with polymer-drugconjugates by dipping the stent in polymer-drug solution or spraying thestent with such a solution. In certain embodiments, suitable materialsfor the implantable device include biocompatible and nontoxic materials,and may be chosen from the metals such as nickel-titanium alloys, steel,or biocompatible polymers, hydrogels, polyurethanes, polyethylenes,ethylenevinyl acetate copolymers, etc. In certain embodiments, theinventive compound is coated onto a stent for insertion into an arteryor vein following balloon angioplasty.

The compounds of this invention or pharmaceutically acceptablecompositions thereof may also be incorporated into compositions forcoating implantable medical devices, such as prostheses, artificialvalves, vascular grafts, stents and catheters. Accordingly, the presentinvention, in another aspect, includes a composition for coating animplantable device comprising a compound of the present invention asdescribed generally above, and a carrier suitable for coating saidimplantable device. In still another aspect, the present inventionincludes an implantable device coated with a composition comprising acompound of the present invention as described generally above, and acarrier suitable for coating said implantable device.

Within other aspects of the present invention, methods are provided forexpanding the lumen of a body passageway, comprising inserting a stentinto the passageway, the stent having a generally tubular structure, thesurface of the structure being coated with (or otherwise adapted torelease) an inventive compound or composition, such that the passagewayis expanded. In certain embodiments, the lumen of a body passageway isexpanded in order to eliminate a biliary, gastrointestinal, esophageal,tracheal/bronchial, urethral and/or vascular obstruction.

In certain embodiments, the invention provides a method of treatment ofany of the disorders described herein, wherein the subject is a human.

In accordance with the foregoing, the present invention further providesa method for preventing or treating any of the diseases or disordersdescribed above in a subject in need of such treatment, which methodcomprises administering to said subject a therapeutically effectiveamount of a compound of the invention or a pharmaceutically acceptablesalt thereof. For any of the above uses, the required dosage will varydepending on the mode of administration, the particular condition to betreated and the effect desired.

Pharmaceutical Compositions

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound of any of the formulae herein (e.g., formula I),or a pharmaceutically acceptable ester, salt, or prodrug thereof,together with a pharmaceutically acceptable carrier.

Compounds of the invention can be administered as pharmaceuticalcompositions by any conventional route, in particular enterally, e.g.,orally, e.g., in the form of tablets or capsules, or parenterally, e.g.,in the form of injectable solutions or suspensions, topically, e.g., inthe form of lotions, gels, ointments or creams, or in a nasal orsuppository form. Pharmaceutical compositions comprising a compound ofthe present invention in free form or in a pharmaceutically acceptablesalt form in association with at least one pharmaceutically acceptablecarrier or diluent can be manufactured in a conventional manner bymixing, granulating or coating methods. For example, oral compositionscan be tablets or gelatin capsules comprising the active ingredienttogether with a) diluents, e.g., lactose, dextrose, sucrose, mannitol,sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum,stearic acid, its magnesium or calcium salt and/or polyethyleneglycol:for tablets also c) binders, e.g., magnesium aluminum silicate, starchpaste, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose and or polyvinylpyrrolidone: if desired d)disintegrants, e.g., starches, agar, alginic acid or its sodium salt, oreffervescent mixtures; and/or e) absorbents, colorants, flavors andsweeteners. Injectable compositions can be aqueous isotonic solutions orsuspensions, and suppositories can be prepared from fatty emulsions orsuspensions. The compositions may be sterilized and/or containadjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressureand/or buffers. In addition, they may also contain other therapeuticallyvaluable substances. Suitable formulations for transdermal applicationsinclude an effective amount of a compound of the present invention witha carrier. A carrier can include absorbable pharmacologically acceptablesolvents to assist passage through the skin of the host. For example,transdermal devices are in the form of a bandage comprising a backingmember, a reservoir containing the compound optionally with carriers,optionally a rate controlling barrier to deliver the compound to theskin of the host at a controlled and predetermined rate over a prolongedperiod of time, and means to secure the device to the skin. Matrixtransdermal formulations may also be used. Suitable formulations fortopical application, e.g., to the skin and eyes, are preferably aqueoussolutions, ointments, creams or gels well-known in the art. Such maycontain solubilizers, stabilizers, tonicity enhancing agents, buffersand preservatives.

Compounds of the invention can be administered in therapeuticallyeffective amounts in combination with one or more therapeutic agents(pharmaceutical combinations). For example, synergistic effects canoccur with other anti-proliferative, anti-cancer, immunomodulatory oranti-inflammatory substances. Where the compounds of the invention areadministered in conjunction with other therapies, dosages of theco-administered compounds will of course vary depending on the type ofco-drug employed, on the specific drug employed, on the condition beingtreated and so forth.

Combination therapy includes the administration of the subject compoundsin further combination with other biologically active ingredients (suchas, but not limited to, a second and different antineoplastic agent) andnon-drug therapies (such as, but not limited to, surgery or radiationtreatment). For instance, the compounds of the invention can be used incombination with other pharmaceutically active compounds, preferablycompounds that are able to enhance the effect of the compounds of theinvention. The compounds of the invention can be administeredsimultaneously (as a single preparation or separate preparation) orsequentially to the other drug therapy. In general, a combinationtherapy envisions administration of two or more drugs during a singlecycle or course of therapy.

In certain embodiments, these compositions optionally further compriseone or more additional therapeutic agents. Alternatively, a compound ofthis invention may be administered to a patient in need thereof incombination with the administration of one or more other therapeuticagents. For example, additional therapeutic agents for conjointadministration or inclusion in a pharmaceutical composition with acompound of this invention may be an approved chemotherapeutic agent, orit may be any one of a number of agents undergoing approval in the Foodand Drug Administration that ultimately obtain approval for thetreatment of protozoal infections and/or any disorder associated withcellular hyperproliferation. In certain other embodiments, theadditional therapeutic agent is an anticancer agent, as discussed inmore detail herein. In certain other embodiments, the compositions ofthe invention are useful for the treatment of protozoal infections. Inthe treatment of cancer or protein degradation disorders, the inventivecompound may be combined with a proteasome inhibitor (e.g., bortezomib,Rl 15777 FTI, MG 132, NPI-0052, etc.). In the treatment of cancer orprotein degradation disorders, the inventive compound may be combinedwith protein degradation inhibitor (e.g. another inventive compound, atubacin-like compound, bortezomib, Rl 15777 FTI, MGl 32, NPI-0052, SAHA,¹⁶⁶Ho-DOTMP, arsenic trioxide, 17 AAG, MG 132, etc.).

It will also be appreciated that the compounds and pharmaceuticalcompositions of the present invention can be employed in combinationtherapies, that is, the compounds and pharmaceutical compositions can beadministered concurrently with, prior to, or subsequent to, one or moreother desired therapeutics or medical procedures. The particularcombination of therapies (therapeutics or procedures) to employ in acombination regimen will take into account compatibility of the desiredtherapeutics and/or procedures and the desired therapeutic effect to beachieved. It will also be appreciated that the therapies employed mayachieve a desired effect for the same disorder (for example, aninventive compound may be administered concurrently with anotheranticancer agent), or they may achieve different effects (e.g., controlof any adverse effects).

The present invention encompasses pharmaceutically acceptable topicalformulations of inventive compounds. The term “pharmaceuticallyacceptable topical formulation,” as used herein, means any formulationwhich is pharmaceutically acceptable for intradermal administration of acompound of the invention by application of the formulation to theepidermis. In certain embodiments of the invention, the topicalformulation comprises a carrier system. Pharmaceutically effectivecarriers include, but are not limited to, solvents (e.g., alcohols, polyalcohols, water), creams, lotions, ointments, oils, plasters, liposomes,powders, emulsions, microemulsions, and buffered solutions (e.g.,hypotonic or buffered saline) or any other carrier known in the art fortopically administering pharmaceuticals. A more complete listing ofart-known carriers is provided by reference texts that are standard inthe art, for example, Remington's Pharmaceutical Sciences, 16th Edition,1980 and 17th Edition, 1985, both published by Mack Publishing Company,Easton, Pa., the disclosures of which are incorporated herein byreference in their entireties. In certain other embodiments, the topicalformulations of the invention may comprise excipients. Anypharmaceutically acceptable excipient known in the art may be used toprepare the inventive pharmaceutically acceptable topical formulations.Examples of excipients that can be included in the topical formulationsof the invention include, but are not limited to, preservatives,antioxidants, moisturizers, emollients, buffering agents, solubilizingagents, other penetration agents, skin protectants, surfactants, andpropellants, and/or additional therapeutic agents used in combination tothe inventive compound. Suitable preservatives include, but are notlimited to, alcohols, quaternary amines, organic acids, parabens, andphenols. Suitable antioxidants include, but are not limited to, ascorbicacid and its esters, sodium bisulfite, butylated hydroxytoluene,butylated hydroxyanisole, tocopherols, and chelating agents like EDTAand citric acid. Suitable moisturizers include, but are not limited to,glycerine, sorbitol, polyethylene glycols, urea, and propylene glycol.Suitable buffering agents for use with the invention include, but arenot limited to, citric, hydrochloric, and lactic acid buffers. Suitablesolubilizing agents include, but are not limited to, quaternary ammoniumchlorides, cyclodextrins, benzyl benzoate, lecithin, and polysorbates.Suitable skin protectants that can be used in the topical formulationsof the invention include, but are not limited to, vitamin E oil,allatoin, dimethicone, glycerin, petrolatum, and zinc oxide.

In certain embodiments, the pharmaceutically acceptable topicalformulations of the invention comprise at least a compound of theinvention and a penetration enhancing agent. The choice of topicalformulation will depend or several factors, including the condition tobe treated, the physicochemical characteristics of the inventivecompound and other excipients present, their stability in theformulation, available manufacturing equipment, and costs constraints.As used herein the term “penetration enhancing agent” means an agentcapable of transporting a pharmacologically active compound through thestratum corneum and into the epidermis or dermis, preferably, withlittle or no systemic absorption. A wide variety of compounds have beenevaluated as to their effectiveness in enhancing the rate of penetrationof drugs through the skin. See, for example, Percutaneous PenetrationEnhancers, Maibach H. I. and Smith H. E. (eds.), CRC Press, Inc., BocaRaton, Fla. (1995), which surveys the use and testing of various skinpenetration enhancers, and Buyuktimkin et al., Chemical Means ofTransdermal Drug Permeation Enhancement in Transdermal and Topical DrugDelivery Systems, Gosh T. K., Pfister W. R., Yum S. I. (Eds.),Interpharm Press Inc., Buffalo Grove, Ill. (1997). In certain exemplaryembodiments, penetration agents for use with the invention include, butare not limited to, triglycerides (e.g., soybean oil), aloe compositions(e.g., aloe-vera gel), ethyl alcohol, isopropyl alcohol,octolyphenylpolyethylene glycol, oleic acid, polyethylene glycol 400,propylene glycol, N-decylmethylsulfoxide, fatty acid esters (e.g.,isopropyl myristate, methyl laurate, glycerol monooleate, and propyleneglycol monooleate) and N-methyl pyrrolidine.

In certain embodiments, the compositions may be in the form ofointments, pastes, creams, lotions, gels, powders, solutions, sprays,inhalants or patches. In certain exemplary embodiments, formulations ofthe compositions according to the invention are creams, which mayfurther contain saturated or unsaturated fatty acids such as stearicacid, palmitic acid, oleic acid, palmito-oleic acid, cetyl or oleylalcohols, stearic acid being particularly preferred. Creams of theinvention may also contain a non-ionic surfactant, for example,polyoxy-40-stearate. In certain embodiments, the active component isadmixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms are made by dissolving or dispensing thecompound in the proper medium. As discussed above, penetration enhancingagents can also be used to increase the flux of the compound across theskin. The rate can be controlled by either providing a rate controllingmembrane or by dispersing the compound in a polymer matrix or gel.

It will also be appreciated that the compounds and pharmaceuticalcompositions of the present invention can be formulated and employed incombination therapies, that is, the compounds and pharmaceuticalcompositions can be formulated with or administered concurrently with,prior to, or subsequent to, one or more other desired therapeutics ormedical procedures. The particular combination of therapies(therapeutics or procedures) to employ in a combination regimen willtake into account compatibility of the desired therapeutics and/orprocedures and the desired therapeutic effect to be achieved. It willalso be appreciated that the therapies employed may achieve a desiredeffect for the same disorder (for example, an inventive compound may beadministered concurrently with another immunomodulatory agent,anticancer agent or agent useful for the treatment of psoriasis), orthey may achieve different effects (e.g., control of any adverseeffects).

For example, other therapies or anticancer agents that may be used incombination with the inventive compounds of the present inventioninclude, but not limited to, surgery, radiotherapy (in but a fewexamples, gamma-radiation, neutron beam radiotherapy, electron beamradiotherapy, proton therapy, brachytherapy, and systemic radioactiveisotopes, to name a few), endocrine therapy, biologic response modifiers(interferons, interleukins, antibodies, aptamers, siRNAs,oligonucletoides, enzyme, ion channel and receptor inhibitors oractivators to name a-few), hyperthermia and cryotherapy, agents toattenuate any adverse effects (e.g., antiemetics), and other approvedchemotherapeutic drugs, including, but not limited to, alkylating drugs(e.g., mechlorethamine, chlorambucil, Cyclophosphamide, Melphalan,Ifosfamide), antimetabolites (e.g., Methotrexate), purine antagonistsand pyrimidine antagonists (e.g., 6-Mercaptopurine, 5-Fluorouracil,Cytarabile, Gemcitabine), spindle poisons (e.g., Vinblastine,Vincristine, Vinorelbine, Paclitaxel), podophyllotoxins (e.g.,Etoposide, Irinotecan, Topotecan), antibiotics (Doxorubicin, Bleomycin,Mitomycin), nitrosoureas (e.g., Carmustine, Lomustine), inorganic ions(e.g., Cisplatin, Carboplatin), enzymes (e.g., Asparaginase), andhormones (e.g., Tamoxifen, Leuprolide, Flutamide, and Megestrol), toname a few. For a more comprehensive discussion of updated cancertherapies see, The Merck Manual, Seventeenth Ed. 1999, the entirecontents of which are hereby incorporated by reference. See also theNational Cancer Institute (NCI) website (www.nci.nih.gov) and the Foodand Drug Administration (FDA) website for a list of the FDA approvedoncology drugs (www.fda.gov/cder/cancer/dmglistfrarne).

In certain embodiments, the pharmaceutical compositions of the presentinvention further comprise one or more additional therapeutically activeingredients (e.g., chemotherapeutic and/or palliative). For purposes ofthe invention, the term “palliative” refers to treatment that is focusedon the relief of symptoms of a disease and/or side effects of atherapeutic regimen, but is not curative. For example, palliativetreatment encompasses painkillers, antinausea medications,anti-pyretics, and anti-sickness drugs. In addition, chemotherapy,radiotherapy and surgery can all be used palliatively (that is, toreduce symptoms without going for cure; e.g., for shrinking tumors andreducing pressure, bleeding, pain and other symptoms of cancer).

The present compounds and compositions can be administered together withhormonal and steroidal anti-inflammatory agents, such as but not limitedto, estradiol, conjugated estrogens (e.g., PREMARIN, PREMPRO, ANDPREMPHASE), 17 beta estradiol, calcitonin-salmon, levothyroxine,dexamethasone, medroxyprogesterone, prednisone, cortisone, flunisolide,and hydrocortisone; non-steroidal anti-inflammatory agents, such as butnot limited to, tramadol, fentanyl, metamizole, ketoprofen, naproxen,nabumetone, ketoralac, tromethamine, loxoprofen, ibuprofen, aspirin, andacetaminophen; anti-TNF-α antibodies, such as infliximab (REMICADE®) andetanercept (ENBREL®).

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of a compound of the present inventionformulated together with one or more pharmaceutically acceptablecarriers. As used herein, the term “pharmaceutically acceptable carrier”means a non-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Thepharmaceutical compositions of this invention can be administered tohumans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), buccally, or as an oral or nasal spray.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, eye ointments, powders and solutionsare also contemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

According to the methods of treatment of the present invention,disorders are treated or prevented in a subject, such as a human orother animal, by administering to the subject a therapeuticallyeffective amount of a compound of the invention, in such amounts and forsuch time as is necessary to achieve the desired result. The term“therapeutically effective amount” of a compound of the invention, asused herein, means a sufficient amount of the compound so as to decreasethe symptoms of a disorder in a subject. As is well understood in themedical arts a therapeutically effective amount of a compound of thisinvention will be at a reasonable benefit/risk ratio applicable to anymedical treatment.

In general, compounds of the invention will be administered intherapeutically effective amounts via any of the usual and acceptablemodes known in the art, either singly or in combination with one or moretherapeutic agents. A therapeutically effective amount may vary widelydepending on the severity of the disease, the age and relative health ofthe subject, the potency of the compound used and other factors. Ingeneral, satisfactory results are indicated to be obtained systemicallyat daily dosages of from about 0.03 to 2.5 mg/kg per body weight (0.05to 4.5 mg/m²). An indicated daily dosage in the larger mammal, e.g.humans, is in the range from about 0.5 mg to about 100 mg, convenientlyadministered, e.g. in divided doses up to four times a day or incontrolled release form. Suitable unit dosage forms for oraladministration comprise from ca. 1 to 50 mg active ingredient.

In certain embodiments, a therapeutic amount or dose of the compounds ofthe present invention may range from about 0.1 mg/kg to about 500 mg/kg(about 0.18 mg/m² to about 900 mg/m²), alternatively from about 1 toabout 50 mg/kg (about 1.8 to about 90 mg/m²). In general, treatmentregimens according to the present invention comprise administration to apatient in need of such treatment from about 10 mg to about 1000 mg ofthe compound(s) of this invention per day in single or multiple doses.Therapeutic amounts or doses will also vary depending on route ofadministration, as well as the possibility of co-usage with otheragents.

Upon improvement of a subject's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease. Thesubject may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

It will be understood, however, that the total daily usage of thecompounds and compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific inhibitory dose for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder, the activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed:the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed; and like factors well known in themedical arts.

The invention also provides for a pharmaceutical combinations, e.g. akit, comprising a) a first agent which is a compound of the invention asdisclosed herein, in free form or in pharmaceutically acceptable saltform, and b) at least one co-agent. The kit can comprise instructionsfor its administration to a subject suffering from or susceptible to adisease or disorder.

The terms “co-administration” or “combined administration” or the likeas utilized herein are meant to encompass administration of the selectedtherapeutic agents to a single patient, and are intended to includetreatment regimens in which the agents are not necessarily administeredby the same route of administration or at the same time.

The term “pharmaceutical combination” as used herein means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g., a compound of the invention and a co-agent, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g., a compound of the invention and a co-agent, are bothadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the twocompounds in the body of the patient. The latter also applies tococktail therapy, e.g., the administration of three or more activeingredients.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, or potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, wool fat, sugars such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes, oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols; such a propyleneglycol or polyethylene glycol; esters such as ethyl oleate and ethyllaurate, agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water, isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. The protein kinaseinhibitors or pharmaceutical salts thereof may be formulated intopharmaceutical compositions for administration to animals or humans.These pharmaceutical compositions, which comprise an amount of theprotein inhibitor effective to treat or prevent a proteinkinase-mediated condition and a pharmaceutically acceptable carrier, areanother embodiment of the present invention.

Examples

The compounds and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not to limit the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the chemicalstructures, substituents, derivatives, formulations and/or methods ofthe invention may be made without departing from the spirit of theinvention and the scope of the appended claims. Definitions of variablesin the structures in schemes herein are commensurate with those ofcorresponding positions in the formulae delineated herein.

Example 1: Synthesis of 1

Synthesis of ethyl 2-(phenylamino)pyrimidine-5-carboxylate

To a solution of compound A in NMP (20 ml) was added aniline (1.0 g,5.36 mmol) and heated at 120° C. for overnight. After the reactioncompleted, the mixture was poured into ice water (20 ml), theprecipitate was collected and washed with methanol (5 ml*2), theprecipitation was isolated by filtration to afford the compound B as apale solid (980 mg, 75%).

Synthesis of N-hydroxy-2-(phenylamino)pyrimidine-5-carboxamide

To a solution of compound B (100 mg, 0.411 mmol) inmethanol/dichloromethane (5/2 ml) was added 98% NH₂OH (1.7 ml, 50% inwater) at 0° C. and stirred for 10 min. A solution of saturated sodiumhydroxide in methanol (1.5 ml) was added and the mixture kept stirringfor another 10 min. The reaction mixture was concentrated in vacuum andthe crude residue was acidified to pH=6-7 with 2N HCl. The precipitatewas collected, washed with water to afford 1 as an off-white solid (90mg, 94.7%).

Example 2: Synthesis of 99

Example 3: Synthesis of 110

Example 4: Synthesis of 111

Example 5: HDAC Enzyme Assays

Compounds for testing were diluted in DMSO to 50 fold the finalconcentration and a ten point three fold dilution series was made. Thecompounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl,0.001% Tween-20, 0.05% BSA, 20 μM TCEP) to 6 fold their finalconcentration. The HDAC enzymes (purchased from BPS Biosciences) werediluted to 1.5 fold their final concentration in assay buffer. Thetripeptide substrate and trypsin at 0.05 μM final concentration werediluted in assay buffer at 6 fold their final concentration. The finalenzyme concentrations used in these assays were 3.3 ng/ml (HDAC1), 0.2ng/ml (HDAC2), 0.08 ng/ml (HDAC3) and 2 ng/ml (HDAC6). The finalsubstrate concentrations used were 16 μM (HDAC1), 10 μM (HDAC2), 17 μM(HDAC3) and 14 μM (HDAC6). Five μl of compounds and 20 μl of enzyme wereadded to wells of a black, opaque 384 well plate in duplicate. Enzymeand compound were incubated together at room temperature for 10 minutes.Five μl of substrate was added to each well, the plate was shaken for 60seconds and placed into a Victor 2 microtiter plate reader. Thedevelopment of fluorescence was monitored for 60 min and the linear rateof the reaction was calculated. The IC₅₀ was determined using Graph PadPrism by a four parameter curve fit.

IC₅₀ (nM) Compound ID HDAC1 HDAC2 HDAC3 HDAC6 2 264 (53.1x) 248 (50x)299 (60.4x) 5 3 210 (26.7x) 347 (44.1x) 354 (44.9x) 8 1 20x 30x 27x 7 4195 (22x) 290 (32.8x) 291 (32.9x) 9 5 247 (16.3x) 395 (26x) 421 (27.8x)15 6 202 (10.1x) 231 (11.6x) 266 (13.3x) 20 7 415 (9.9x) 629 (14.9x) 606(14.4x) 42 15 1410 (102.2x) 1390 (100.8x) 1852 (134.3x) 14 8 82 (2.1x)82 (2.1x) 219 (5.7x) 38 16 332 (15.7x) 384 (18.1x) 600 (28.3x) 21 17 182(33.5x) 161 (29.6x) 198 (36.5x) 5 18 136 (10.8x) 170 13.5x) 177 (14.1x)13 10 329 (32.6x) 631 (62.5x) 430 (42.6x) 10 12 212 (14.1x) 453 (30.2x)352 (23.5x) 15 20 10598 (12.3x 17328 (20.1x) 16328 (18.9x) 864 21 241(46x) 526 (100.3x) 484 (92.2x) 5 22 222 (83.1x) 446 (166.7x) 313 (117x)3 19 251 (69.1x) 478 (131.7x) 319 (88x) 4 23 155 (60.4x) 343 (134x) 257(100.2x) 3 24 133 (26x) 261 (50.9x) 193 (37.6x) 5 25 516 (50.9x) 938(92.6x) 830 (81.9x) 10 26 159 (31.4x) 345 (68.3x) 270 (53.4x) 5 27 417(63.5x) 700 (106.6x) 745 (113.5x) 7 28 111 (30.7x) 187 (51.5x) 181(50.1x) 4 29 464 (60.9x) 808 (106x) 520 (68.2x) 8 30 88 (27.5x) 181(56.5x) 141 (44.1x) 3 31 206 (19.8x) 362 (34.7x) 221 (21.1x) 10 32 47(22.6x) 83 (39.8x) 85 (41x) 2 33 110 (56.4x) 226 (116.2x) 169 (86.7x) 234 20 (15.5x) 28 (21.5x) 34 (25.7x) 1 35 77 (42.3x) 140 (76.2x) 128(7.0x) 2 IC50 (nM) Compound ID HDAC1 HDAC2 HDAC3 HDAC6 36 40 (17.5x) 71(31.2x) 75 (33x) 2 37 89 (17.8x) 127 (25.4x) 149 (29.8x) 5 38 470(67.5x) 720 (103.5x) 891 (128.1x) 7 39 263 (62.6x) 387 (92.2x) 495(117.9x) 4 40 38 (19.8x) 56 (28.7x) 70 (36.2x) 2 41 214 (48.9x) 259(59.2x) 326 (74.5x) 4 42 78 (19.5x) 97 (24.1x) 130 (32.3x) 4 43 90(22.8x) 120 (30.3x) 107 (27.1x) 4 44 173 (15.9x) 253 (23.2x) 307 (28.2x)11 45 377 (106.9x) 523 (148.5x) 506 (143.4x) 4 46 40 (21.4x) 53 (28.4x)58 (31.2x) 2 47 223 (55.8x) 346 (86.7x) 300 (75.1x) 4 48 363 (48.8x) 601(80.8x) 401 (53.9x) 7 49 219 (99.5x) 338 (154x) 303 (138.1x) 2 50 1166(288.1x) 1516 (374.6x) 2214 (547.1x) 4 51 468 (41.2x) 624 (55x) 382(33.6x) 11 52 185 (60.9x) 248 (81.6x) 254 (83.6x) 3 53 24 (14.9x) 30(19.2x) 43 (27.2x) 2 54 1370 (64.4x) 2105 (98.9x) 1568 (73.7x) 21 55 460(60.2x) 587 (76.8x) 631 (82.6x) 8 57 526 (86x) 713 (116.6x) 901 (147.4x)6 58 552 (60.8x) 701 (77.2x) 1085 (119.5x) 9 59 607 (15.3x) 815 (20.5x)873 (22x) 40 60 129 (53.5x) 142 (58.9x) 237 (98.1x) 2 61 1336 (35.9x)1540 (41.4x) 3263 (87.7x) 37 62 35 (22.5x) 56 (36x) 55 (35.2.x) 2 631445 (77.6x) 1798 (96.5x) 1746 (93.7x) 19 65 1128 (61x) 1701 (91.9x)7482 (404.4x) 19 66 39 (29.1x) 39 (29.2x) 47 (35.2x) 1 67 61 (26.1x) 79(33.8x) 100 (42.8x) 2 68 761 (254.8x) 1083 (362.4x) 1480 (495.3x) 3 69204 (41.5x) 240 (48.7x) 299 (60.9x) 5 70 645 (217.2x) 972 (327.2x) 1313(441.9x) 3 71 500 (202.9x) 912 (370x) 1260 (511.4x) 2 72 5752 (306.9x)8006 (427.2x) 12297 (656.2x) 19 73 94 (60x) 128 (81.9x) 219 (139.5x) 274 889 (147.5x) 1015 (168.4x) 2376 (394.2x) 6 75 1142 (374.2x) 966(316.3x) 1882 (616.6x) 3 76 624 (153.3x) 609 (149.7x) 1058 (260x) 4 77328 (74x) 340 (76.9x) 434 (98.1x) 4 78 598 (137.3x) 669 (153.6x) 1327(304.6x) 4 79 1268 (221.6x) 1830 (319.8x) 4676 (817.2x) 6 80 85 (40.4x)127 (60.2x) 212 (100.3x) 2 81 47 (18.3x) 78 (30.2x) 120 (46.4x) 3 82 16(9.9x) 18 (11.2x) 22 (13.6x) 2 83 447 (139.2x) 452 (140.9x) 928 (289x) 384 2226 (302.2x) 2971 (403.4x) 4515 (613x) 7 85 67 (18.1x) 96 (26.2x)135 (36.9x) 4 86 788 (60.3x) 1292 (98.9x) 2891 (221.2x) 13 87 85 (45.4x)116 (61.6x) 233 (124.1x) 2 88 711 (215.2x) 978 (296.1x) 2243 (679.1x) 389 223 (64.4x) 270 (78.1x) 549 (158.8x) 3 90 27 (19.9x) 36 (26.2x) 41(30.2x) 1 91 160 (72.3x) 288 (130.2x) 374 (169.4x) 2 92 1359 (41.3x)2235 (68x) 4422 (134.4x) 33 93 80 (25.9x) 107 (34.7x) 157 (51x) 3 94 22(12x) 32 (17.4x) 50 (27.4x) 2 95 586 (176.7x) 722 (217.7x) 2272 (685x) 396 2341 (271.8x) 2465 (286.2x) 5709 (662.8x) 9 97 19 (8.3x) 28 (12.1x)37 (16.4x) 2 98 147 (39.9x) 239 (64.8x) 4252 (115.2x) 4 99 200 (47.3x)320 (75.6x) 345 (81.5x) 4 100 3206 (339.9x) 4761 (504.7x) 13474(1428.4x) 9 101 2123 (283.5x) 2570 (343.2x) 11223 (1498.8x) 7 102 13(7.4x) 21 (11.3x) 33 (18x) 2 103 578 (12.6x) 832 (18.1x) 1098 (23.9x) 46104 813 (16.7x) 1174 (24.1x) 1172 (24x) 49 105 214 (54.5x) 361 (92.1x)361 (92x) 4 106 92 (25.3x) 133 (36.5x) 174 (47.7x) 4 107 60 (2.7x) 9(3.7x) 19 (8x) 2 108 219 (42.1x) 313 (60.3x) 355 (68.3x) 5 109 324(79.5x) 385 (94.3x) 843 (206.7x) 4 110 1409 (34.5x) 1631 (39.9x) 3460(84.7x) 41 111 911 (48x) 767 (40.5x) 1327 (70x) 19 120 38 (9.8x) 53(13.5x) 128 (32.8x) 4 121 27 (12.4x) 32 (14.5x) 99 (44.8x) 2 122 893(44.8x) 898 (45x) 3944 (197.8x) 20 123 13 (4.8x) 12 (4.3x) 26 (9.6x) 3124 5 (3.6x) 6 (3.9x) 8 (5.9x) 1 125 18 (8.7x) 19 (9x) 57 (27.1x) 2 1267 (3.2x) 9 (3.9x) 22 (9.8x) 2 127 50 (23.9x) 57 (27.1x) 119 (56.1x) 2128 16 (8.1x) 17 (8.9x) 49 (24.8x) 2 129 43 (12.7x) 52 (15.4x) 124(36.6x) 3 130 140 (43.3x) 159 (48.9x) 521 (160.9x) 3 131 17 (4.8x) 19(5.5x) 62 (18x) 3 132 95 (24.4x) 96 (24.4x) 316 (80.8x) 4 133 594 (188x)483 (152.9x) 727 (229.8x) 3 134 47 (14x) 40 (11.8x) 99 (29.2x) 3 135 149(22.4x) 118 (17.7x) 344 (51.8x) 7 136 45 (13.3x) 40 (11.9x) 107 (31.4x)3 137 27 (7.1x) 21 (5.5x) 70 (18.5x) 4 138 50 (6.2x) 49 (6x) 113 (13.9x)8 139 386 (90.9x) 479 (112.8x) 782 (184x) 4 140 55 (8x) 62 (9x) 128(18.5x) 7 141 13 (3.2x) 17 (4.2x) 54 (13x) 4 142 17 (3x) 23 (4x) 64(11x) 6 143 12 (3.4x) 17 (5x) 29 (8.4x) 4 144 25 (7.9x) 34 (10.8x) 58(18.3x) 3 145 104 (18.1x) 135 (23.5x) 384 (66.6x) 6 146 16 (5.2x) 22(7x) 57 (18.6x) 3 147 64 (18.2x) 73 (20.9x) 189 (54x) 3 148 583 (182.9x)615 (193.1x) 2157 (677.2x) 3 149 339 (80.1x) 330 (78x) 1324 (312.9x) 4150 320 (66.1x) 330 (68.4x) 1777 (367.7x) 5 151 584 (176.6x) 476(143.8x) 1373 (414.9x) 3 152 248 (11.4x) 220 (10.1x) 649 (29.9x) 4 153528 (64.4x) 491 (59.9x) 1255 (153.1x) 8 154 1372 (263.9x) 1600 (307.8x)3365 (647.4x) 5 155 262 (37.9x) 231 (34x) 1219 (176x) 7

INCORPORATION BY REFERENCE

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference. Unless otherwisedefined, all technical and scientific terms used herein are accorded themeaning commonly known to one with ordinary skill in the art.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended with be encompassed by the following claims.

1. A compound of formula I:

or a pharmaceutically acceptable salt, ester or prodrug thereof,wherein, the ring comprising X₁, X₂, X₃, and X₄ is selected from thefollowing:

R₁ is H, optionally substituted alkyl, optionally substituted aryl,optionally substituted arylalkyl optionally substituted heteroarylalkyl,or optionally substituted heteroaryl; and R₂ is H or alkyl; wherein R₁and R₂ are both not simultaneously H. 2-4. (canceled)
 5. The compound ofclaim 4, wherein R₁ is H, methyl, ethyl, propyl, i-propyl, butyl,i-butyl, t-butyl, pentyl, hexyl, phenyl, benzyl, phenethyl, naphthyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, piperidinyl,pyrrolidinyl, tetrahydropyranyl, thiophenyl, or tetrahydrofuranyl, eachof which may be optionally substituted.
 6. The compound of claim 5,wherein R₁ is substituted with 1-4 groups independently selected fromoptionally substituted alkyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocycloalkyl, halo,haloalkyl, alkoxy, haloalkoxy, and nitro.
 7. The compound of claim 6,wherein R₁ is substituted with 1-4 groups independently selected frommethyl, phenyl, pyridyl, morpholino, indolyl, pyrazinyl, F, Cl, Br,methoxy, OCF₃, and trifluoromethyl; each of which may be furthersubstituted by alkyl, aryl, haloalkyl, haloalkoxy, halo, or alkoxy.8-16. (canceled)
 17. The compound of claim 1, of formula III:

or a pharmaceutically acceptable salt, ester or prodrug thereof,wherein, R₂ is H or methyl; R₁ is H, methyl, ethyl, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, piperidinyl, pyrrolidinyl,tetrahydropyranyl, tetrahydrofuranyl, benzyl, phenethyl, pyridyl, orphenyl, each of which may be optionally substituted with 1-4 groupsindependently selected from methyl, phenyl, pyridyl, morpholino,indolyl, pyrazinyl, F, Cl, Br, methoxy, OCF₃, trifluoromethyl, OH, NH₂,CN, and NO₂, each of which may be further substituted by alkyl, aryl,haloalkyl, halo, or alkoxy. 18-21. (canceled)
 22. The compound of claim17, wherein R₁ is phenyl or pyridyl.
 23. (canceled)
 24. The compound ofclaim 22, wherein R₁ is further substituted with 1-4 substituentsindependently selected from methyl, F, Cl, OCH₃, CF₃, OCF₃, phenyl,pyridyl, morpholinyl, or NO₂.
 25. The compound of claim 1, of formulaIV:

or a pharmaceutically acceptable salt, ester or prodrug thereof,wherein, R₂ is H or methyl; R_(x) is H, alkyl, haloalkyl, alkenyl, aryl,arylalkyl, heteroarylalkyl, heteroaryl, heterocyclic, carbocyclic,—C(O)R₅, or —S(O)_(p)R₅; each of which may be optionally substituted;R_(y) is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted; or R_(x) and R_(y) together withthe carbon to which each is attached, forms a cycloalkyl orheterocycloalkyl, each of which is optionally substituted; each R_(A) isindependently alkyl, alkoxy, cycloalkyl, aryl, heterocycloalkyl,heteroaryl, arylalkyl, heteroarylalkyl, haloalkyl, haloalkoxy, halo, OH,—NO₂, —CN, or —NH₂; or two R_(A) groups together with the atoms to whicheach is attached, can form an optionally substituted ring; each R₅ isindependently H, alkyl, carbocycle, heterocycle, aryl, or optionallysubstituted heteroaryl; m is 0, 1, 2, 3, or 4; and p is 0, 1, 2, or 3.26-37. (canceled)
 38. The compound of claim 1, of formula VI:

or a pharmaceutically acceptable salt, ester or prodrug thereof,wherein, R_(x) is H, alkyl, or aryl; each of which is optionallysubstituted; R_(y) is H or alkyl; each of which is optionallysubstituted; each R_(A) is independently alkyl, alkoxy, cycloalkyl,aryl, heterocycloalkyl, heteroaryl, arylalkyl, heteroarylalkyl,haloalkyl, haloalkoxy, halo, OH, —NO₂, —CN, or —NH₂; and m is 0, 1, or2.
 39. The compound of claim 38, wherein R_(x) is H, methyl, ethyl, orphenyl.
 40. The compound of claim 38, wherein R_(y) is H, methyl orethyl.
 41. The compound of claim 38, wherein m is 1 or 2, and each R_(A)is independently methyl, phenyl, F, Cl, methoxyl, or CF₃.
 42. (canceled)43. The compound of claim 1, of formula VII-A:

or a pharmaceutically acceptable salt, ester or prodrug thereof,wherein, ring A is a heteroaryl or heterocyclic; R₂ is H or methyl;R_(x) is H, alkyl, haloalkyl, alkenyl, aryl, arylalkyl, heteroarylalkyl,heteroaryl, heterocyclic, carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each ofwhich may be optionally substituted; R_(y) is H, alkyl, haloalkyl,alkenyl, aryl, arylalkyl, heteroarylalkyl, heteroaryl, heterocyclic,carbocyclic, —C(O)R₅, or —S(O)_(p)R₅; each of which may be optionallysubstituted; or R_(x) and R_(y) together with the carbon to which eachis attached, form a cycloalkyl or heterocycloalkyl, each of which isoptionally substituted; each R_(A) is independently alkyl, alkoxy,cycloalkyl, aryl, heterocycloalkyl, heteroaryl, arylalkyl,heteroarylalkyl, haloalkyl, haloalkoxy, halo, OH, —NO₂, —CN, or —NH₂;each R₅ is independently H, alkyl, carbocycle, heterocycle, aryl, oroptionally substituted heteroaryl; and m is 0, 1, 2, 3, or
 4. 44. Thecompound of claim 43, wherein ring A is acridinyl, azocinyl,benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl,benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl,NH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isochromanyl,isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl,isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl,oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl; 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl,phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl,phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4thiadiazolyl, thianthrenyl,thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl,thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl,1,2,5-triazolyl, 1,3,4-triazolyl, or xanthenyl.
 45. (canceled)
 46. Thecompound of claim 43, wherein R_(x) is H, alkyl, aryl, or heteroaryl.47. The compound of claim 43, wherein R_(y) is H, alkyl, aryl, orheteroaryl.
 48. The compound of claim 43, wherein R_(x) is H, methyl orpyridine, and R_(y) is H.
 49. The compound of claim 43, wherein R_(x)and R_(y) together with the carbon to which each is attached, forms acyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, orcyclooctyl, each of which is optionally substituted.
 50. The compound ofclaim 49, wherein R_(x) and R_(y) together with the carbon to which eachis attached, forms tetrahydropyran, piperidine, piperazine, morpholine,tetrahydrofuran, tetrahydrothiophene, pyrrolidine, oxozolidine, orimidazolidine, each of which is optionally substituted.
 51. The compoundof claim 43, wherein R_(A) is H, alkyl, alkoxy, cycloalkyl, aryl,heterocycloalkyl, heteroaryl, arylalkyl, heteroarylalkyl, haloalkyl,haloalkoxy, halo, OH, —NO₂, —CN, or —NH₂; and m is 0 or
 1. 52-56.(canceled)
 57. The compound of claim 1, selected from the following:


58. (canceled)
 59. A pharmaceutical composition comprising a compound ofclaim 1, or a pharmaceutically acceptable ester, salt, or prodrugthereof, together with a pharmaceutically acceptable carrier.
 60. Amethod of selectively inhibiting HDAC6 over other HDACs in a subject,comprising administering to the subject a compound of claim 1 or apharmaceutically acceptable salt, ester or prodrug thereof. 61-63.(canceled)
 64. A method of treating a disease mediated by HDAC6 in asubject comprising administering to the subject a compound of claim 1 ora pharmaceutically acceptable salt, ester or prodrug thereof. 65-74.(canceled)